JP2023098990A - Expression of Recombinant Proteins in Trichoplusia ni Pupae - Google Patents

Abstract

To provide means and methods to increase the efficiency of recombinant protein expression, relating to the industrial production of recombinant proteins in insect pupae; and to provide pupae itself comprising baculovirus, and pupae infected, transformed, transduced or transfected with baculovirus or bacmid.SOLUTION: A pupa comprises recombinant baculovirus and/or bacmid derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the pupa belonging to the genus Trichoplusia, Rachipulsia, Spodoptera, Heliothis or Helicoverpa.SELECTED DRAWING: None

Description

The present invention may be included in the field of biotechnology and in particular relates to the industrial production of recombinant proteins in insect pupae, especially Trichoplusia ni (T. ni) pupae. includes means and methods for increasing the efficiency of expression of (including optimizing its industrial production). Still further, the present invention relates to the pupae themselves containing baculovirus, pupae infected with, transformed, transduced or transfected with, baculovirus or bacmid.

The baculovirus expression vector system (BEVS) is an established method for the production of recombinant proteins, such as those used as vaccines, therapeutic molecules or diagnostic reagents. Due to its overexpression potential and rapid development, BEVS is one of the most attractive options for the production of any recombinant protein of interest. The industry's most adopted baculovirus vector for recombinant protein expression is Autographa californica nuclear polyhedrosis using Spodoptera frugiperda 9 (Sf9) or 21 (Sf21) insect cells as the preferred expression host. Viruses (Autographa california)
nica multinuclear polyhedrosis virus) (AcMNPV) (Non-Patent Document 1), and insect larvae of Trichoplusia ni (T. ni) as living biofactories (Non-Patent Document 2)
It is based on Since BEVS was developed in the 80's (3), it has successfully produced hundreds of recombinant proteins ranging from cytosolic enzymes to membrane-bound proteins in baculovirus-infected insect cells.

Recently, new baculovirus vectors have been described. For example, US Pat. Nos. 6,301,000 and 6,000,000 describe recombinant DNA elements for expression of recombinant proteins in insects and insect cells.

Approximately 70,000 tons of silk are produced globally each year in the process of converting the low-value raw material, mulberry leaves, into the high-value protein-based product, silk. Insects are very efficient protein producers due to their accelerated metabolism. Bombyx mori (B. mori, silkworm), or T. mori Lepidoptera, such as di (cabbage looper), are two of the most used insects in biotechnology. They increase in size about 5000-fold in less than 2 weeks and B. Each harpoon insect produces more than one kilometer of silk.
Cells derived from the silk gland can produce about 80 μg of protein per cell per day, whereas the best mammalian cell culture systems produce only about 50 pg of protein per cell per day.

Insects as living biofactories therefore constitute a promising alternative to insect cells, traditional fermentation techniques, and plant-derived proteins due to their production versatility, scalability, automatability, efficiency and speed of development. For example, insects as living biofactories circumvent the need for bioreactors for protein expression in eg insect cells. Bioreactors are inefficient, expensive, technically complex (takes years to build, are difficult to validate, require highly skilled individuals to operate, are prone to contamination, and are unreliable. is a technical and economic barrier to the production of new and existing recombinant proteins. Furthermore, bioreactors face the problem of limited scalability.

B. Fox larvae are widely used as a living biofactory for recombinant protein expression using a baculovirus expression vector system (Non-Patent Document 4, Non-Patent Document 5
, Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9).

Also, T. The larvae of Di. Document 16, Non-Patent Document 17).

In silkworms, comparative studies demonstrated that larvae instead of pupae gave the highest expression yields for most proteins (18, 19). Also, a decrease in pupal susceptibility to baculovirus infection in silkworms was described in relation to pupal age (Non-Patent Document 20). In addition, larvae are generally orally infected with baculovirus instead of inoculation (injection) for scale-up of production. Since pupae cannot be infected orally, they must be injected manually, a tedious and laborious process. In addition, silkworm pupae are covered with a thick cocoon that must be removed (manually) in a generally tedious process before virus inoculation takes place.

Due to the lower protein expression yield in common silkworm pupae and the difficulty of its manipulation,
The use of larvae for recombinant protein production has become universally preferred.

Similar disadvantages are seen in other systems such as Hyalophora cecropia pupae. H. Expression of specific proteins in pupae of T. cecropia lower than that of larvae (Non-Patent Document 21). In addition, the Hyalophora cecropia moth is difficult to keep, is strictly monogamous (one generation per year), produces very few eggs per cycle, and has a dense cocoon (no virus inoculation). The thick cocoons must be removed (by hand) before it can be done, which is generally a labor intensive task). All of these disadvantages make the Hyalophora cecropia pupae a poor system for the expression of recombinant proteins, particularly a system that is not efficient, scalable and automated for the expression of recombinant proteins.

There is a need to make systems more efficient and easier to automate (scale up) for the expression of recombinant proteins in insects using BEVS, particularly for the industrial expression of recombinant proteins in insects using BEVS.

After intensive studies, the inventors have found a solution to the above problem, i.e. a more efficient than larval expression in pupae belonging to the order Lepidoptera, more preferably to the species Trichoplusia ni.
Furthermore, it allows almost complete automation (scale-up), especially on an industrial scale, increasing efficiency and
A protein expression system has been found that reduces the costs associated with recombinant protein expression.

WO2012/168493 International Publication No. 2012168492

Nettleship, J.E., Assenberg, R., Diprose, J.M., Rahman-Huq, N., Owens, R.J. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. 2010, 172, 55 -65 Gomez-Casado E, Gomez-Sebastian S, Nunez MC, Lasa-Covarrubias R, Martinez-Pulgarin S, Escribano JM. Insect larvae biofactories as a platform for influenza vaccine production. Protein Expr Purif. 79: 35-43. 2011 Smith, G.E., M.D. Summers, and M.J. Fraser. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. Wang, DN; Liu, JW; Yang, GZ; Zhang, WJ; Wu, XF. 2002. Cloning of anti-LPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro. 1), 1-7 Wu, XF; Kamei, K; Sato, H; Sato, S; Takano, R; Ichida, M; Mori, H; silkworm (Bombyx mori L.) using recombinant baculovirus. Protein Expression And Purification, 21(1), 192-200 Kulakosky, PC; Hughes, PR; Wood, HA. 1998. N-linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells. Glycobiology, 8(7), 741-745 Suzuki, T; Kanaya, T; Okazaki, H; Ogawa, K; Usami, A; Watanabe, H; Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene. Journal Of General Virology, 78, 3073-3080 Sumathy, S; Palhan, VB; Gopinathan, KP. 1996. Expression of human growth hormone in silkworm larvae through recombinant Bombyx mori nuclear polyhedrosis virus. Protein Expression And Purification, 7(3), 262-268 U. Reis, B. Blum, B.U. von Specht, H. Domdey, J. Collins, Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus, Biotechnology (NY) 10 (1992) 910-912 Perez-Martin, E., Gomez-Sebastian, S., Argilaguet, J.M., Sibila, M., Fort, M., Nofrarias, M., Kurtz, S., Escribano, J.M., Segales, J., Rodriguez, F. ., 2010. Immunity conferred by an experimental vaccine based on the recombinant PCV2 Cap protein expressed in Trichoplusia ni-larvae. Vaccine 28 (11), 2340-2349 Medin, JA; Hunt, L; Gathy, K; Evans, RK; Coleman, MS. 1990. Efficient, low-cost protein factories - expression of human adenosine-deaminase in baculovirus-infected insect larvae. of the United States of America, 87(7), 2760-2764 Shafer, AL; Katz, JB; Eernisse, KA. 1998. Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Diseases, 42(1), 28-34 Cha, HJ; Pham, MQ; Rao, G; Bentley, WE. 1997. Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. Windass, JD; Suner, MM; Cory, JS. 2000. Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni Journal of Invertebrate Pathology, 75(3), 226-236 Cubillos, C.; Angulo, I.; Barderas, M. G.; Barcena, J.; Escribano, J. M. 2007. Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV. Virology, 364(2), 422-430 Perez-Filgueira, D. A.; Gonzalez-Camacho, F.; Gallardo, C.; Resino-Talavan, P.; Blanco, E.; validation of recombinant serological tests for African swine fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae. Journal of Clinical Microbiology, 44(9), 3114-3121 Hellers, M; Gunne, H; Steiner, H. 1991. Expression and posttranslational processing of preprocecropin-a using a baculovirus vector. European Journal of Biochemistry, 199(2), 435-439 Akihiro Usami et al. Hidekazu Nagaya, Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm, J. Biochem. 2011;149(2):219-227 Chazarra S, Aznar-Cervantes S, Sanchez-del-Campo L, Cabezas-Herrera J, Xiaofeng W, Cenis JL, Rodriguez-Lopez JN, Purification and kinetic properties of human recombinant dihydrofolate reductase produced in Bombyx mori chrysalides, Appl Biochem Biotechnol. 2010 Nov;162(7):1834-46 Journal of General Virology (1992), 73, 3195-320 Hellers, M. and Steiner, H.; Insect Biochem. Molec. Biol., Vol 22, Bo.1, pp. 35-39, 1992

Accordingly, the present invention provides baculovirus derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to produce recombinant proteins for use in diagnostics, vaccines and therapeutic treatments. It relates to the use of pupae (chrysalises) belonging to the order Lepidoptera, more preferably belonging to the species Trichoplusia ni, in combination with a viral vector. The use of pupae from Trichoplusia ni species for recombinant protein expression, in particular the industrial use of pupae from Trichoplusia ni species for recombinant protein expression, has not yet been reported.

The present invention relates to Autographa californica multicapsid nuclear polyhedrosis virus (Ac
pupae containing recombinant baculoviruses and/or transfer vectors/bacmids derived from MNPV) are provided.

Furthermore, the present invention allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. and a recombinant homology region (
hr) are provided.

The invention also relates to the use of pupae of the invention for the expression of recombinant proteins.

Furthermore, the present invention provides
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) inoculating the pupae of step (a) with a recombinant baculovirus derived from
(c) for a period of time sufficient to express the at least one recombinant protein (
incubating the inoculated pupae of b);
(d) obtaining pupae containing at least one recombinant protein;
(e) optionally harvesting at least one recombinant protein;
(f) optionally purifying at least one recombinant protein;
A method is provided for producing at least one recombinant protein comprising

Further, herein:
(a) preparing a pupa contained in a cocoon;
(b) treating the pupae contained in the cocoons with a solution of a salt of hypochlorous acid, preferably sodium hypochlorite, preferably by specially designed equipment;
(c) obtaining silk-free (and optionally essentially externally sterilized) pupae;
Methods are provided that can be automated to reduce manipulations for producing silk-free pupae belonging to the order Lepidoptera, comprising:

A method of producing a recombinant baculovirus comprising:
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
transfecting the pupae of step (a) with a suitable transfer vector/baculovirus to produce recombinant baculovirus derived from PV);
(c) incubating the inoculated pupae of step (b) for a period of time sufficient to produce recombinant baculovirus;
(d) obtaining pupae containing the recombinant baculovirus;
(e) optionally harvesting the recombinant baculovirus;
(f) optionally purifying the recombinant baculovirus;
including.

The present invention also provides a precision pump, a mobile mechanical arm, and a Lepidoptera, preferably Trichoplusia, Rachiplusia, Spodopter.
a) Genus Heliothis, Manduca, Helicover
pa) genus Ascalapha or Samia, more preferably Trichoplusia, Rachiprusia, Spodoptera, Heliotis or Helicovelpa, even more preferably Trichoplusia ni Seed, Rachiprussia Nu (Rachi
plusia nu) species, Spodoptera frugiperda species, Heliothis virescens species, Helicoverpa
armigera species, Helicoverpa Zea species, Mand
a (movable) needle(s) suitable for injecting fluids into pupae belonging to the species Ascalapha odorata, Ascalapha odorata, or Samia cynthia; It relates to an apparatus comprising

Large-scale disposable T.I. Fig. 12 is a diagram of an insect rearing module; Fig. 5: 5th instar larvae growing in an insect rearing module. FIG. 10 compares cocoons formed by Bombyx mori and Trichoplusia dilepidoptera (left part of image) and pupae without silk (right part of image). T. Fig. 2 is a diagram of a semi-automatic device for the removal of silk from the cocoons of D.; 1 is a schematic representation of a machine comprising two containers and one mechanical arm in which disposable rearing modules are arranged; The first container contains hypochlorous acid and a system for releasing liquid through the rearing module containing the cocoons, which helps dissolve the silk surrounding the pupae more efficiently. A second container is for washing the pupae, which sprays the pupae with water. The lid of this container has a system of providing air to dry the pupae. At the end of the process, the pupae are silk-free and ready to be used for infection or refrigerated until use. FIG. 2 is a diagram of the baculovirus expression cassette used for the production of green fluorescent protein (GFP). A) A diagram of a conventional baculovirus expression cassette using the polyhedrin promoter (eg SEQ ID NO:23). B) Ac-ie-01 cDNAs encoding transactivators IE1 and IE0 expressed under the control of the polyhedrin promoter; enhancer sequence hr1 and chimeric promoter p6.9-p10 driving expression of GFP ( FIG. 3 is a diagram of a TB expression cassette comprising, eg, SEQ ID NO: 17 and a nucleic acid sequence encoding GPF, eg, SEQ ID NO: 38). Robotic T. cerevisiae injecting recombinant baculovirus Fig. 2: Automatic inoculation of two pupae. A) Schematic representation of the inoculation robot. B) Schematic representation of the matrix of ostia in which insect pupae are placed. These stomas containing pupae have perforated lids, are stackable, facilitate transport of pupae to production laboratories, and are compatible with inoculation robots. In the same panel, the actual photographic image of the ostium containing the pupa and including the operculum is shown. C) Schematic representation of pupae inoculation with a needle connected to a robotic arm. FIG. 4 is a comparative analysis of the expression yield of GFP protein in infected pupae by using conventional baculovirus TB(−) (polyhedrin promoter, eg SEQ ID NO: 23) or TB-modified baculovirus TB(+). . A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae infected with TB(-) or TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantitation of GFP production yield obtained in pupae infected with each baculovirus analyzed, expressed in mg/g pupal biomass. FIG. 2 is a diagram of baculovirus expression cassettes used to produce capsid protein (Cap) from porcine circovirus type 2 (eg SEQ ID NO: 26) or hemagglutinin (HA) from influenza virus (eg SEQ ID NO: 30). A) A diagram of a conventional baculovirus expression cassette using the polyhedrin promoter. B) Ac-ie-01 cDNA encoding transactivators IE1 and IE0 expressed under the control of the polyhedrin promoter; enhancer sequence hr1 and chimeric promoter p6 driving expression of the above mentioned proteins. TB expression cassette containing .9-p10. Infected pupae by using conventional baculovirus TB(-) (polyhedrin promoter, SEQ ID NO: 10, SEQ ID NO: 28) or TB modified baculovirus TB(+) (eg SEQ ID NO: 27 or SEQ ID NO: 29) FIG. 2 is a comparative analysis of the expression yield of Cap protein (eg SEQ ID NO: 26) in . A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae infected with TB(-) or TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantitation of Cap production yield obtained in pupae infected with each baculovirus analyzed, expressed in mg/g pupal biomass. of HA protein (SEQ ID NO: 30) in infected pupae by using conventional baculovirus TB(−) (polyhedrin promoter, SEQ ID NO: 10) or TB-modified baculovirus TB(+) (eg SEQ ID NO: 31) FIG. 10 is a comparative analysis of expression yields. A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae infected with TB(-) or TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of HA production yields obtained in pupae infected with each baculovirus analyzed, expressed in mg/g pupal biomass. T. used to produce GFP, Cap (SEQ ID NO: 26), HA (SEQ ID NO: 30), and VP60 proteins from rabbit hemorrhagic disease virus (RHDV, SEQ ID NO: 32 or SEQ ID NO: 33) in larvae and pupae of D. FIG. 3 is a diagram of a baculovirus expression cassette. Ac-ie-01 cDNAs encoding transactivators IE1 and IE0 expressed under the control of the polyhedrin promoter; enhancer sequence hr1 and chimeric promoter p6.9 driving expression of the proteins mentioned above. Schematic representation of the TB expression cassette containing -p10. FIG. 10 is a comparative analysis of the expression yield of GFP protein in infected pupae and larvae. A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae (P) or larvae (L) infected with TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of GFP production yields obtained with infected pupae or larvae, expressed in mg/g insect biomass. FIG. 10 is a comparative analysis of Cap protein expression yield in infected pupae and larvae. A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae (P) or larvae (L) infected with TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of Cap production yields obtained with infected pupae or larvae, expressed in mg/g insect biomass. FIG. 10 is a comparative analysis of HA protein expression yield in infected pupae and larvae. A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae (P) or larvae (L) infected with TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of HA production yields obtained with infected pupae or larvae, expressed in mg/g insect biomass. Comparative analysis of the expression yield of RHDV capsid VP60 proteins (G1 and RHDVb) in infected pupae and larvae. A) Coomassie blue staining of SDS-PAGE separating protein extracts from pupae (P) or larvae (L) infected with TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of RHDV capsid protein production yields obtained in infected pupae or larvae, expressed in mg/g insect biomass (L) with TB(+) baculovirus. (-) corresponds to extracts from uninfected control pupae. B) Quantification of RHDV capsid protein production yields obtained in infected pupae or larvae, expressed in mg/g insect biomass. TB (+) baculovirus expressing VP60 proteins from RHDV G1 and RHDVb. FIG. 12. VLPs formed after infection of two pupae. Extracts from pupae infected with each baculovirus at the optimal production time were processed for VLP purification. Samples were observed by electron microscopy using negative staining. The figure shows VLPs at two different magnifications. VLPs obtained from the two baculoviruses displayed identical size and shape. Photomicrographs are representative of the fields analyzed. FIG. 1 is a schematic example of a procedure for obtaining a viral inoculum from infected pupae in the absence of insect cell culture. Schematic example of upstream and downstream processing procedures for obtaining purified recombinant protein from infected pupae in three steps. A) T. FIG. 2 is a diagram of the production of the pupae, their manipulation and optional transport to the final destination (eg, pharmaceutical company) for recombinant protein production. B) Preservation of pupae, which can be stored for several months before processing in situ or easily transported to other locations for further downstream processing, using the device of the present invention. FIG. 10. Robotic inoculation of recombinant baculovirus, incubation, and freezing of insect biomass. C) Illustration of downstream processing of frozen biomass by conventional means, including homogenization, tangential flow filtration, and protein purification. FIG. 2 is a comparison of the expression yield of Cap protein of porcine circovirus in insect cells by using conventional baculovirus and TopBac-modified baculovirus in insect cells and insect pupae. FIG. 2 is a schematic example of the procedure for obtaining viral inoculum from infected pupae. FIG. 2 is a schematic example of a downstream processing procedure to obtain purified recombinant protein from infected pupae.

DEFINITIONS As used herein, "pupa" refers to the life stage of an insect that undergoes metamorphosis. The pupal stage is found in insects that undergo holometabolism (holometabolous insects). These insects go through four life stages: embryo, larva, pupa and adult. Butterfly pupae are also called chrysalis. insects are
The pupae may be protected by a cocoon, a silken casing that protects the pupae of many insects.

As used herein, "baculovirus" refers to a family of viruses that are infectious to invertebrates (primarily infecting insects and arthropods). "Recombinant Baculovirus"
contain recombinant DNA that has been further introduced through, for example, homologous recombination or gene transposition. The recombinant baculovirus may be derived from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV).

As used herein, an "expression cassette" includes recombinant DNA elements involved in the expression of a particular gene, such as the gene itself and/or elements (eg promoters) that control the expression of this gene. For example, an expression cassette useful in the present invention contains the following recombinant DNA elements.
1. a nucleic acid sequence (at least a promoter) that allows for and preferably controls expression of a recombinant protein, such as those described herein below, and
2. normal of said regulator obtained during baculovirus infection of insect cells or
That is, nucleic acid sequences that allow expression of baculovirus transcriptional regulators such as IE-1 and IE-0 above endogenous levels.

In some embodiments, the expression cassette further comprises an enhancer homology region (hr), such as hr1, operably linked to the promoter of the sequence encoding the recombinant protein. The recombinant DNA element forming part of an expression cassette of the invention may be present in a single nucleic acid molecule. The recombinant DNA element forming part of the expression cassette may be present in different nucleic acid molecules. It is preferred that the different nucleic acid molecules are present within the same cell.

As used herein, "recombinant DNA" is an artificial DNA in which DNAs that do not normally occur together are combined by manipulation by the combination or insertion of one or more DNA strands.
refers to the form of

As used herein, "recombinant DNA element" refers to a functional element within recombinant DNA such as a promoter, enhancer or gene.

A "transcription regulator" as used herein is, for example, by binding to enhancer or repressor regions and/or recruiting additional proteins involved in transcription.
Refers to regulatory proteins that have the ability to regulate the transcription of specific genes. IE-1 and its splice variant IE-0 are transcriptional regulators endogenously expressed during baculovirus infection. Expression levels of protein IE-1, protein IE-0 and/or fragments thereof are determined at both the mRNA and protein levels by methods conventionally known to those skilled in the art such as quantitative PCR or Western blot analysis. obtain.

According to the present invention, IE-1, IE-0 and/or fragments thereof may be recombinantly expressed to increase the total level of these proteins over endogenous levels during baculovirus infection. This includes, for example, introducing a further copy of the endogenous gene,
or by manipulating the expression of the endogenous gene's promoter. Additional copies of the endogenous gene can be introduced as a transgene under the control of a suitable promoter such as polh or _pB29 .

IE-1, IE-0 and fragments thereof can be encoded by the nucleic acids of SEQ ID NO: 1 (also called Ac-ie-01) through SEQ ID NO:5. SEQ ID NO: 1 is IE-1 and I
SEQ ID NO:2 is the coding sequence (CDS) for IE-1 and SEQ ID NO:3 is the CDS for IE-0. SEQ ID NO:4 and SEQ ID NO:5 are the CDSs of the N-terminal domains of IE-1 and IE-0, respectively, which retain catalytic transcriptional regulatory activity. The proteins encoded by SEQ ID NOs:2-5 are represented by SEQ ID NOs:6-9, respectively.

Nucleic acid and amino acid sequences referred to in the present invention each have the default parameters of E
Identified using the MBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emb
oss_needle/) shall be distinguished from other nucleic acid and amino acid sequences by their degree of sequence identity or sequence similarity. Methods for generating such variants include random or site-directed mutagenesis, site saturation mutagenesis, PCR-based fragment assembly, DNA shuffling, in vitro or in vivo homologous recombination, and methods of gene synthesis. is mentioned.

As used herein, a "variant" is a nucleic acid or amino acid sequence that differs from a parent nucleic acid or amino acid sequence at one or more positions, the difference being the addition of a nucleic acid or amino acid residue. , deletions and/or substitutions.

As used herein, a "region of homology" (hr) is an incomplete 30 bp near their center.
It is composed of repeat units of approximately 70 bp with a palindrome of . For example, the region of homology is repeated at 8 locations in the AcMNPV genome with 2-8 repeats on each side.
Homologous regions are implicated as both transcriptional enhancers and baculovirus DNA replication origins.

As used herein, an "enhancer region" refers to a regulatory sequence that increases transcription levels of the associated gene upon binding of a transcriptional regulator.

As used herein, "recombinant protein" refers to a protein produced from recombinant DNA. Such proteins can be used for human and animal benefit, industrially
It may have commercial or therapeutic uses.

As used herein, "operably linked" refers to two nucleic acid sequences that are joined such that one affects the other, eg, in terms of transcriptional regulation.

As used herein, "promoter" refers to a DNA sequence capable of initiating transcription upon binding of RNA polymerase. The sequences may further contain binding sites for various proteins that regulate transcription, such as transcription factors. The promoter sequence may be composed of different promoter fragments (either different or identical fragments) that are closely located in the DNA sequence and may be separated by linkers or spacers. Such promoters are called chimeric promoters.

As used herein, a "transfer vector" is a vector (ie, a DNA molecule used as a vehicle to hold genetic material) that allows the insertion of genetic information into the baculovirus genome.

As used herein, "bacmid" refers to a plasmid construct containing DNA sequences sufficient to produce a baculovirus when transfected into a cell or insect.

As used herein, "cloning vector" refers to any vector suitable for cloning and generally contains restriction sites, an origin of replication for bacterial growth, and the presence of a selectable marker.

The cloning vectors that can be used in the present invention include (i) sequences for expression above endogenous levels of protein IE-0, protein IE-1 and/or fragments thereof, as well as (iii) driving expression of the recombinant protein. (ii) a recombined homology region (hr) linked to a promoter suitable for For example, a cloning vector may contain a nucleic acid sequence encoding a recombinant protein (ie, a cloning vector containing an expression cassette, also called a "donor vector"). Alternatively, the cloning vector lacks such sequences.

A "vaccine" as used herein may preferably be defined as a biological preparation containing a recombinant protein that provides active acquired immunity against a particular disease.

As used herein, the term "about" means ±1% of the stated value, or "about"
The term means ±2% of the stated value, or the term “about” means ±5% of the stated value, or the term “about” means ±10% of the stated value. or the term "about" means the indicated value ± 20% of the value, or the term "about" means the indicated value ± 30%, preferably the term "about" is exactly Means indicated value (±0%).

DETAILED DESCRIPTION The present invention provides pupae containing recombinant baculoviruses and/or transfer vectors/bacmids. The present invention surprisingly provides the introduction of a recombinant baculovirus, particularly a sequence causing expression of a baculovirus transcriptional regulator above endogenous levels, into insect pupae,
and optionally the introduction of enhancer homology region (hr) sequences, promoters or combinations of promoters can increase the production of recombinant proteins to unprecedented levels. This is particularly important for the expression of recombinant proteins in vivo.
The utility of this system for industrial production of recombinant proteins in vivo is demonstrated.

Pupa of the Invention The invention provides a pupa comprising a recombinant baculovirus, and/or a transfer vector, and/or a bacmid. The recombinant baculovirus and/or transfer vector and/or bacmid are preferably derived from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The pupa is of the order Lepidoptera, preferably of the genera Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa, more preferably Trichoplusia ni spp., Rachiprusia nu spp., Spodoptera frugiperda spp., Heliothis virescens spp., Helicoverpa armigella spp., Helicovelpa zi spp., Manzuka sexta spp., Ascarafa odorata spp., Or it preferably belongs to Samia cynthia species. Even more preferably, the pupa belongs to the Trichoplusia ni species. In a preferred embodiment, the pupae of the invention do not belong to the Bombyx mori species. In a preferred embodiment, the pupae of the invention do not belong to the species Hyalophora cecropia.

In a preferred embodiment, the pupae of the invention are Trichoplusia, preferably Trichoplusia, comprising a recombinant baculovirus and/or transfer vector/bacmid derived from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV).・It is a pupa belonging to two species.

The pupae of the present invention, especially T. The second pupa offers several advantages for the expression of recombinant proteins, especially in an automated and scalable process. For example, T. The second pair of moths is 1
They may have about 1000 eggs per cycle. Furthermore, T. The cocoons produced by the pupae of A. niger are not as thick as the cocoons that cover the pupae of other species (e.g. Bombycus mori, or Hyalophora cecropia), thus allowing automated and scalable production processes (industrial production of recombinant proteins). ) for the use of pupae in T. Pupae of two are particularly suitable. Therefore, preferably Autographa californica multicapsid nuclear polyhedrosis virus (AcMNP
V) a recombinant baculovirus and/or a transfer vector derived from V), and/
or bacmid-infected T. The pupa of D. ni provides an efficient, scalable, and easily automated system for recombinant protein production. Further advantages of this system are its high production capacity (up to 20 times more productive than bioreactors), the fact that it is technically simple and easy to implement and validate, reduced costs (fixed capital investment for the use of bioreactors). more than 90% reduction in viral load), lower cost of goods, faster generation (baculovirus system), higher efficiency of difficult-to-produce proteins, and higher quality and safety of manufactured products.

The inventors of the present application have surprisingly found that pupae, in particular of the order Lepidoptera, preferably of the genus Trichoplusia,
Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa, more preferably Trichoplusia ni , Rachiprusia nu, Spodoptera frugiperda, Heliothis virescens, Helicovelpa armigera, Helicovelpa zi, Manzuka sexta, Ascarafa
We found that the expression of the recombinant protein in pupae belonging to the species Odorata or Samia cynthia was comparable and even higher than in larvae.

A patent application published as WO 2005/010002 discloses recombinant baculovirus and transfer vectors and bacmids that can be included in the pupae of the present invention.

Preferably, the recombinant baculovirus and/or transfer vector and/or bacmid contained in the pupae according to the invention may contain recombinant DNA. For example, the recombinant baculovirus and/or transfer vector and/or bacmid contained in the pupae according to the invention comprise a nucleic acid sequence encoding a recombinant protein, the recombinant protein being a subunit monomeric vaccine, a subunit multivalent selected from the group consisting of body vaccines, virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP) Preferably, and/or the recombinant protein is not a protein endogenously produced by the pupa, as described below. For example, recombinant baculoviruses and/or transfer vectors contained in pupae according to the invention, and/
Alternatively, the bacmid allows expression above endogenous levels of proteins IE-1, protein IE-0, and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. (e.g., expression above endogenous levels includes additional copies of nucleic acid sequences that allow expression of protein IE-1, protein IE-0, and/or fragments thereof in recombinant baculoviruses). can be obtained by existence). Recombinant baculoviruses and/or transfer vectors and/or bacmids contained in pupae according to the present invention contain a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein. is preferably further included.

The transfer vectors that can be included in the pupae of the present invention contain (i) sequences for expression above endogenous levels of protein IE-0, protein IE-1, and/or fragments thereof, in addition to (iii) It preferably contains (ii) a recombined homology region (hr) linked to a suitable promoter to drive expression. In one preferred aspect, the transfer vector comprises a nucleic acid sequence encoding said recombinant protein, while in another preferred embodiment the transfer vector lacks such sequences. In preferred embodiments, the transfer vector is a bacmid.

Transfer vectors and/or bacmids are commercially available baculovirus expression systems “Bac-to-Bac™” (Invitrogen™), “BacPA
K™” (Clontech™), “FlashBAC™” (Oxford Expressio
Technologies™), “BacuVance™” (GenScript™),
"Bac-N-Blue DNA™" (invitrogen™), "BaculoD
irect(TM)" (invitrogen(TM)), "BacVector(TM)" 100
0, 2000, 3000 (Novagen™), "DiamondBac™" (Sig
ma-Aldrich™), or “BaculoGold™” (BD biosciences™).

Pupa of the present invention (preferably from the order Lepidoptera, preferably from the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably from the genus Trichoplusia, Rachiprusia, Spodoptera) , Heliothis or Helicovelpa, more preferably Trichoplusia ni spp., Rachiprusia nu spp., Spodoptera frugiperda spp., Heliothis virescens spp., Helicoverpa armigera spp., Helicovelpa zi spp., Manzuka sexta spp., Ascarafa・ Odrata species,
or Samia cynthia species, or other Lepidoptera susceptible to AcMNPV, even more preferably belonging to the genus Trichoplusia and the species Trichoplusia ni) exceeding the endogenous levels obtained during baculovirus infection. The protein IE- which functions as a transcriptional regulator
1, protein IE-0, and/or fragments thereof that allow expression above endogenous levels. The recombinant DNA elements described above are preferably introduced into pupae by recombinant baculovirus.

Nucleic acid sequences allowing expression of protein IE-1, protein IE-0 and/or fragments thereof functioning as transcriptional regulators above the endogenous levels obtained during baculovirus infection according to the invention are
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
is preferably selected from the group consisting of

the sequences of variants of SEQ ID NO: 1 to SEQ ID NO: 5 are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85% relative to the sequences of SEQ ID NO: 1 to SEQ ID NO: 5, More preferably at least 90%, and most preferably at least 95% identical.

the sequences of the variants of SEQ ID NO: 6 to SEQ ID NO: 9 are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85% relative to the sequences of SEQ ID NO: 6 to SEQ ID NO: 9, More preferably at least 90%, and most preferably at least 95% similar.

The pupae of the invention further comprise a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein, and/or a recombinant baculovirus, and/or nucleic acid sequence. It may further comprise a transfer vector and/or a bacmid.

The recombination homology region (hr) is preferably the sequence (hr1) shown in SEQ ID NO:21.

The promoter driving the expression of the recombinant protein is
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
is preferably selected from the group of nucleic acids comprising

In a preferred embodiment, a recombinant promoter, a sequence encoding a transcriptional regulator, and
A nucleic acid sequence (nucleic acid sequence contained in the pupae of the present invention) comprising a combination of an enhancer region is
Selected from the group consisting of SEQ ID NO:15-SEQ ID NO:20.

The recombinant promoter, transcription regulator-encoding sequence, and enhancer region of the invention need not form part of a single molecule; instead, these sequences are operatively linked. ie, form part of different molecules as long as they are contained within the same cell within the pupa.

The pupae and/or recombinant baculoviruses and/or transfer vectors and/or bacmids of the invention may further comprise a nucleic acid sequence encoding a recombinant protein. This nucleic acid sequence comprises a nucleic acid sequence allowing expression above endogenous levels of protein IE-1, protein IE-0, and/or fragments thereof, and optionally a homologous region (h
r), the sequences of which are described above.

Recombinant proteins include subunit monomer vaccines, subunit multimer vaccines,
It is preferably selected from the group consisting of virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents and green fluorescent protein (GFP). .

In preferred embodiments, the recombinant protein is a virus-like particle protein, preferably selected from the group consisting of:
(a) a capsid protein from porcine circovirus, preferably porcine circovirus type 2 (e.g. SEQ ID NO: 26);
(b) foot and mouth disease virus VP1 protein, VP3 protein or VP0 protein;
(c) canine parvovirus VP1 and VP2 proteins;
(d) porcine parvovirus VP1 and VP2 proteins;
(e) human norovirus (genogroup I or II) capsid proteins;
(f) a calicivirus capsid protein;
(g) L derived from human papillomavirus, preferably human papillomavirus 16
1 protein,
(h) hepatitis E protein E2;
(i) Infectious bursal disease virus VP1 protein, VP2 protein and V
P3 protein,
(j) an astrovirus ORF2-encoded protein;
(k) influenza virus HA protein (e.g. SEQ ID NO: 30), NA protein and M1 protein;
(l) hepatitis B core antigen and surface antigen;
(m) rabbit calicivirus, preferably rabbit hemorrhagic disease virus RHDVb and R
VP60 proteins derived from HDVG1 (e.g. SEQ ID NO:32 and SEQ ID NO:33);
(n) Human parvovirus VP1 and VP2 proteins.

For example, the recombinant protein may be:

A capsid protein from a porcine circovirus, preferably porcine circovirus type 2, for example represented by the amino acid sequence of SEQ ID NO:26 or encoded by the nucleic acid sequence of SEQ ID NO:25.

Foot and mouth disease virus (FMDV) VP1, VP3 and VP0 proteins, the sequences of which are shown, for example, in or may be derived from the following sequences:
- FMDV serotype O whole genome: GenBank JX570650.1
-FMDV serotype A whole genome: GenBank HQ832592.1
- FMDV serotype C whole genome: GenBank AY593810.1
- FMDV serotype SAT1 whole genome: GenBank AY593846.1
- FMDV serotype SAT2 whole genome: GenBank JX014256.1
- FMDV serotype ASIA1 whole genome: GenBank HQ631363.1.

Canine parvovirus VP1 and VP2 proteins, the sequences of which are shown, for example, in or may be derived from the following sequences:
- canine parvovirus VP1 gene for capsid protein VP1, partial cds, strain: 1887/f/3. GenBank: AB437434.1.
- canine parvovirus VP1 gene for capsid protein VP1, partial cds, strain: 1887/M/2. GenBank: AB437433.1.
- canine parvovirus VP2 gene, all cds, strain: HNI-2-13. GenBank
: AB120724.1.
- canine parvovirus VP2 gene, all cds, strain: HNI-3-4. GenBank:
AB120725.1.
- canine parvovirus VP2 gene, all cds, strain: HNI-3-11. GenBank
: AB120726.1.
- canine parvovirus VP2 gene, all cds, strain: HNI-4-1. GenBank:
AB120727.1.
- canine parvovirus VP2 gene, all cds, strain: HNI-1-18. GenBank
: AB120728.1.
- canine parvovirus VP2 protein (VP2) gene, all cds. GenBank:
DQ354068.1.
- canine parvovirus VP2 gene, all cds, strain: HCM-6. GenBank: AB
120720.1.
- canine parvovirus isolate Taichung VP2 gene, all cds. GenBan
k: AY869724.1.
- canine parvovirus VP2 gene, all cds, strain: HCM-8. GenBank: AB
120721.1.
- canine parvovirus type 1 proteins VP1 and VP2: GenBank AB5188
83.1
- canine parvovirus type 2a VP1 and VP2. GenBank: M24003.1
- canine parvovirus type 2b VP2: GenBank FJ005265.1
- canine parvovirus type 2C VP2: GenBank FJ005248.1

Porcine parvovirus VP1 and VP2 proteins, the sequences of which are shown, for example, in or may be derived from the following sequences:
- porcine parvovirus strain 693a. GenBank: JN400519.1
- porcine parvovirus strain 8a. GenBank: JN400517.1

Human parvovirus VP1 and VP2 proteins, the sequences of which are shown, for example, in or may be derived from the following sequences:
- human parvovirus strain B19 VP1 full cds. GenBank: AF264149.1
- human parvovirus B19 isolate Vn115 NS1 (NS1), 7.5 kDa protein (NS1), VP1 (VP1), 9.5 kDa protein (VP1) and VP2 (
VP2) gene, all cds. GenBank: DQ357065.1
- Genes of B19 virus isolates FoBe VP1 (VP1) and VP2 (VP2), all cds. GenBank: AY768535.1
- B19 virus isolates Br543 NS1 (NS1), VP1 (VP1) and VP2 (
VP2) gene, all cds. GenBank: AY647977.1
- Genes of human parvovirus 4 isolate VES065CSF NS1, VP1 and VP2, all cds. GenBank: HQ593532.1
- human parvovirus 4 isolate VES085CSF NS1 gene, partial cds; and VP1 and VP2 genes, full cds. GenBank: HQ593531.1
- Parvovirus B19 strain BB-2 NS1, VP1 and VP2 genes, all cds. G.
enBank: KF724387.1

A human norovirus (genogroup I or II) capsid protein, the sequence of which, for example, is shown in or may be derived from the following sequences:
- Norwalk virus: GenBank M87661, NP056821
- Southampton virus: GenBank L07418
- Mexican virus: GenBank U22498
- Setovirus: GenBank AB031013
- Cibavirus: GenBank AB042808
- Rosedale virus: GenBank X86557
- Snow Mountain virus: GenBank U70059
- Hawaii virus: GenBank U07611

Rabbit hemorrhagic disease virus VP60 protein, the sequence of which is shown, for example, in or may be derived from the following sequences:
- RHDV AST/89 Whole Genome: GenBank: Z49271.2
- RHDV N11 whole genome: GenBank: KM878681.1
- RHDV CBVal16 whole genome; GenBank: KM979445.1
- SEQ ID NO:32
- SEQ ID NO:33

Human papillomavirus L1 protein, the sequence of which is shown in or may be derived from, for example, the following sequences:
- HPV6: GenBank: JN252323.1
- HPV11: GenBank: JQ773411.1
- HPV16: GenBank DQ155283.1
- HPV18: GenBank FJ528600.1

Hepatitis E virus E2 protein, the sequence of which is shown, for example, in or may be derived from the following sequences:
- Hepatitis E virus, Whole Genome NCBI Reference Sequence: NC_001434.1
- porcine hepatitis E virus isolate ITFAE11 capsid protein gene. GenBan
k: JN861806.1

VP1, VP2 and VP3 proteins of infectious bursal disease virus
The protein, the sequence of which may be shown in or derived from, for example, the following sequences:
- Infectious bursal disease virus VP1 (VP1) gene, all cds. GenBan
k: AY099457.1
- Infectious bursal disease virus isolate PT VP1 gene, all cds. GenBa
nk: DQ679814.1
- Infectious bursal disease virus isolate OE/G2 VP1 gene, all cds. Ge
nBank: DQ679813.1
- Infectious bursal disease virus isolate OA/G1 VP1 gene, all cds. Ge
nBank: DQ679812.1
- Infectious bursal disease virus isolate HOL VP1 gene, all cds. GenB
ank: DQ679811.1
- Infectious bursal disease virus strain TL2004 VP1 gene, all cds. Gen
Bank: DQ118374.1
- Infectious bursal disease virus isolate CA-K785 VP1 gene, all cds.
GenBank: JF907705.1
- Infectious bursal disease virus isolate D495 VP1 gene, all cds. Gen
Bank: JF907704.1
- Infectious bursal disease virus strain A-BH83 VP1 mRNA, total cds. G.
enBank: EU544149.1
- Infectious bursal disease virus strain Cro-Pa/98 VP1 gene, all cds.
GenBank: EU184690.1
- Infectious bursal disease virus VP2 mRNA, total cds. GenBank: A
Y321509.1
- Infectious bursal disease virus VP2 gene, VP3 gene, VP4 gene, all c
ds. GenBank: M97346.1
- Infectious bursal disease virus VP2 gene, all cds. GenBank: AF5
08177.1

calicivirus capsid protein, the sequence of which is shown, for example, in the sequence below,
or may be derived from:
- feline calicivirus capsid protein gene, all cds. GenBank: L097
19.1
- feline calicivirus capsid protein gene, all cds. GenBank: L097
18.1
- human calicivirus HU/NLV/Wort against capsid protein (ORF2)
ley/90/UK RNA, strain HU/NLV/Wortley/90/UK. GenB
ank: AJ277618.1
- human calicivirus HU/NLV/This against capsid protein (ORF2)
tlehall/90/UK RNA, strain HU/NLV/Thistlehall/90
/UK. GenBank: AJ277621.1
- human calicivirus HU/NLV/Vale against capsid protein (ORF2)
tta/95/Malta RNA, strain HU/NLV/Valetta/95/Malt
a. GenBank: AJ277616.1
- human calicivirus NLV/Stav/95/Nor capsid protein gene, all c
ds. GenBank: AF145709.1
- bovine intestinal calicivirus strain Bo/CV500-OH/2002/US capsid protein gene, all cds. GenBank: AY549155.1
- human calicivirus NLV/Mora/97/SE capsid protein gene, all cd
s. GenBank: AY081134.1
- Human calicivirus NLV/Potsdam 196/2000/DE capsid protein gene, all cds. GenBank: AF439267.1
- human calicivirus NLV/1581-01/SWE capsid protein gene, all c
ds. GenBank: AY247442.1
- Human calicivirus, Hu/NLV/GII/MD134-10/1987/US capsid protein gene, all cds. GenBank: AY030313.1

Astrovirus ORF2-encoding protein, the sequence of which is shown in or may be derived from, for example, the following sequences:
- Porcine Astrovirus 4 ORF1b gene, partial cds; and ORF2 gene, full c
ds. GenBank: JX684071.1
- Astrovirus MLB1 HK05, whole genome. NCBI Reference Sequence: NC_0143
20.1
- Astrovirus wild boar/WBAstV-1/2011/HUN, whole genome. NCBI Reference Sequence: NC_016896.1
- Human Astrovirus BF34, whole genome. NCBI Reference Sequence: NC_024472.
1
- Astrovirus MLB1 strain Hu/ITA/2007/PR326/MLB1 RNA
dependent RNA polymerase (ORF1b) gene, partial cds; and capsid protein (ORF2) gene, full cds. GenBank: KF417713.1
- human astrovirus 5 strain Hu/Budapest/HUN5186/2012/HU
N nonstructural protein (ORF1a) and nonstructural protein (ORF1b) genes, partial cds; and capsid protein (ORF2) gene, full cds. GenBan
k: KF157967.1.
- Human Astrovirus 1 isolate Shanghai capsid protein (ORF2) gene, all cds. GenBank: FJ792842.1
- Human Astrovirus type 8 orf2 gene for capsid protein. GenBan
k: Z66541.1

Influenza virus HA, NA and M1 proteins, the sequences of which are shown in or may be derived from, for example, the following sequences:
- SEQ ID NO:30
- Influenza A virus against hemagglutinin (A/duck/Chiba/25
-51-14/2013 (H7N1)) HA gene, all cds. GenBank: AB8
13060.1
- synthetic construct hemagglutinin (HA) mRNA, total cds. GenBank: DQ868
374.1
- influenza virus A (A/swine/Shandong/2/03 (H5N1
)) Hemagglutinin (HA) gene, all cds. GenBank: AY646424.1
- cDNA coding for influenza A HA. GenBank: E01133.1
- Influenza A virus (A/swine/Korea/S452/2004 (H9
N2)) NA gene, all cds. GenBank: AY790307.1
- Influenza A virus (A/Thailand/2(SP-33)/2004(H
5N1)) Neuraminidase (NA) gene, all cds. GenBank: AY5551
52.3
- Influenza A virus against neuraminidase (A/swine/Binh D
oung/02_16/2010 (H1N2)) NA gene, all cds. GenBank
: AB628082.1
- Influenza A virus (A/chicken/Jalgaon/8824/200
6(H5N1)) neuraminidase (NA) gene, all cds. GenBank: DQ8
87063.1
- Influenza A virus SC35M M2 and M1 genes, all cds. Gen
Bank: DQ266100.1
- Influenza virus type/Leningrad/134/47/57 (H2N2) M
1 and M2 RNA, total cds. GenBank: M81582.1
- Influenza A virus SC35M M2 and M1 genes, all cds. Gen
Bank: DQ266100.1
- matrix protein 2, influenza A virus against matrix protein 1 (A/Toshigi/2/2010 (H1N1)) M2 gene, M1 gene, all cds
. GenBank: AB704481.1

Hepatitis B core and surface antigens, the sequences of which are shown, for example, in or may be derived from the following sequences:
- Hepatitis B virus strain HBV248 precore and core protein genes, all cds. GenBank: KP857118.1
- Hepatitis B virus strain HBV401 precore and core protein genes, total cds. GenBank: KP857113.1
- Hepatitis B virus strain HBV403 precore and core protein genes, all cds. GenBank: KP857068.1
- hepatitis B virus S gene for hepatitis B surface antigen, partial cds, isolate: B050
3327 (PTK). GenBank: AB466596.1

Preferably, the recombinant protein is not a protein endogenously produced by the pupa. For example, a recombinant protein is not a protein endogenously produced by a pupa, such as a pupa of the species Hyalophora cecropia. For example, the recombinant protein is not Cecropin A or attacin.

The pupae of the present invention may be silk-free pupae or may be inside a silk cocoon. If the pupae are not silk-free pupae, those skilled in the art know how to remove the cocoon silk. For example, the cocoon filaments are preferably treated with a salt, preferably a salt of hypochlorous acid (HClO), preferably a solution of sodium hypochlorite (NaClO) (herein referred to as a "dissolving solution").
n)”). As shown in FIG. 3, this procedure may be automated by specially designed equipment.

Use of the Pupae of the Invention for Expression of Recombinant Proteins The pupae of the invention, in any variant thereof, can be used for the expression of recombinant proteins. Accordingly, the present invention provides the use of a pupa of the invention for the expression of a recombinant protein, preferably a recombinant protein as detailed herein above.

Methods of Producing Recombinant Proteins of the Invention The present invention also provides methods of producing recombinant proteins (preferably not endogenously produced by the pupa, as already described above). for example,
A method of obtaining at least one recombinant protein according to the invention comprises:
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) inoculating the pupae of step (a) with a recombinant baculovirus derived from
(c) for a period of time sufficient to express the at least one recombinant protein (
incubating the inoculated pupae of b);
(d) obtaining pupae containing at least one recombinant protein;
(e) optionally harvesting at least one recombinant protein;
(f) optionally purifying at least one recombinant protein;
comprising or consisting of

The pupae of step (a) of the above method may preferably be pupae according to the invention, as described in detail above. The pupae of step (a) preferably belong to the order Lepidoptera. The pupa belongs to the order Lepidoptera,
preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa or Samia, preferably Trichoplusia,
Rachiprusia, Spodoptera, Heliothis or Helicovelpa, more preferably Trichoplusia ni spp., Rachiprusia nu spp., Spodoptera frugiperda spp., Heliothis virescens spp., Helicoverpa armigera spp., Helicovelpa spp. It preferably belongs to the species Sexta, Ascarafa odorata, or Samia cynthia, even more preferably Trichoplusia ni. As already described above, the pupae may contain nucleic acid sequences allowing expression of the recombinant protein. As already described above, the pupae endogenously contain protein IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. It may also contain nucleic acid sequences that allow expression above sexual levels (see above "Pupae of the Invention").

The pupae of step (a) above may be developed from eggs. For example, the pupae of step a) above are
For example, eggs may be allowed to develop from egg to pupa for 15 to 18 days in specially designed disposable or reusable incubators. The pupae of step a) may also be provided already in pupae form.

If the pupa of step (a) above is inside a cocoon, the method of producing the recombinant protein of the invention may further comprise removing the cocoon filaments from the pupa. For example, cocoon filaments are preferably added to the It is preferably soluble in a solution of a salt containing hypochlorous acid (HClO), preferably sodium hypochlorite (NaClO). For example, 0.1% to 5% pupae
The pupae may be immersed in a solution of sodium hypochlorite at a weight/volume concentration or sprayed with the solution. Dissolution of cocoon filaments may take several seconds to several minutes. The selection of silk from the pupae in step (a) is preferably carried out in semi-automated or automated form (robotic silk selection) when the pupae are inside the cocoon. In this regard, the fact that the cocoons of some species, such as Hyalophora cecropia or Bombycus mori, are thick cocoons, is a significant advantage for other genera or species (such as Trichoplusia, particularly Trichoplusia ni). disadvantageous. Pupae of the genus Trichoplusia, particularly Trichoplusia ni, have silky cocoons that are not very dense and can be easily dissolved. This represents the advantage that the entire process can be performed in an automated or semi-automated fashion, thus increasing efficiency and lowering overall costs.

The cocoon may comprise a recipient comprising a dissolution liquid applied to the cocoon, preferably by compressed air turbulence, to reduce the time required for dissolution of the silk cocoon.
It can be removed with a semi-automatic machine. The silk-free pupae may then be washed in a wash container to remove traces of lysate and then air dried.

Therefore, silk-free pupae are preferred, as it is easier to follow step (b). Therefore, pupae with very dense cocoons are less preferred. For example, pupae of Bombyx harpoon have very dense, thick, dense cocoons, and as described above, for several minutes
It cannot be easily removed by dissolving with a salt solution. The same is true for the pupae of Hyalophora cecropia. Bombyx mori and/or Hyalophora cecropia pupal cocoons must be removed manually.

On the contrary, T. The pupae of other species, such as C. japonicus, contain a less dense silk cocoon and can be easily dissolved as described above. Therefore, these pupae are preferred for the method of producing recombinant proteins of the invention, since their cocoons can be removed by automated or semi-automated procedures, and are injected with recombinant baculovirus (step (b)). Facilitates scale-up for obtaining ready-made pupae.

After removing the filament, the pupae are preferably washed with water to remove traces of salt solution (eg sodium hypochlorite). The silk-free pupae are then dried and before performing step (b),
It may be stored at low temperature (eg 4°C). For example, silk-free pupae may be stored at a low temperature (eg, 4° C.) for up to one month before performing step (b).

Therefore, the present invention also provides
(a) providing a pupa contained in a cocoon (preferably a pupa of the invention as described above);
(b) treating said pupae contained in cocoons with a salt solution, preferably a hypochlorous acid salt, preferably a sodium hypochlorite solution, as described in detail above;
(c) obtaining silk-free (preferably essentially sterile) pupae;
A method for producing a silk-free pupa (preferably a silk-free pupa belonging to Lepidoptera, preferably a pupa of the present invention) comprising:

Accordingly, the present invention also provides pupae of the invention that are essentially silk-free (ie, cocoon-free). In a preferred embodiment, the essentially silk-free pupae of the present invention do not belong to the Bombyx mori species. In a preferred embodiment, the essentially silk-free pupae of the present invention do not belong to the species Hyalophora cecropia. In a preferred embodiment, the essentially silk-free pupae of the present invention are of the genera Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia. , Rachiprusia, Spodoptera, Heliotis, or Helicovelpa. In a preferred embodiment, the essentially silk-free pupae of the present invention are Trichoplusia ni sp., Rachiprusia nu sp., Spodoptera frugiperda sp., Heliothis virescens sp., Helicovelpa armigella sp. It belongs to the species Manzuka sexta, Ascarafa odorata, or Samia cynthia, more preferably Trichoplusia ni.

Step (b) of the method of the present invention comprises: step (a) with a recombinant baculovirus derived from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV);
concerning the inoculation of pupae. Recombinant baculoviruses that can be contained in pupae of the invention are described in detail above and an exemplary schematic is shown in FIG. 4B. Therefore, in step (b) it is preferred to inoculate the pupae of the invention with a recombinant baculovirus to provide pupae according to the invention comprising a recombinant baculovirus as described above. The patent application published as WO 2005/010000 discloses a recombinant baculovirus that can be inoculated into pupae according to the invention in step (b) of a method for producing a recombinant protein of the invention. A recombinant baculovirus comprises a nucleic acid sequence (expression cassette) encoding a recombinant protein, preferably a recombinant protein selected from the group defined herein above.

Silk-free pupae are preferred as subsequent inoculation with recombinant baculovirus is easier and can be automated or semi-automated, facilitating scale-up and reproducibility of the method.

Inoculation of recombinant baculovirus according to step (b) can be performed by techniques known in the art to those skilled in the art. "Inoculation", as defined herein, is the introduction of a substance into the body, in this case the introduction of recombinant baculovirus into the pupa. Inoculation may also be referred to as "injection". Because the larvae are inoculated with baculovirus, this process is also sometimes referred to herein as "infection of larvae with baculovirus."

Said inoculation is preferably carried out by injecting the pupae with an amount of solution containing at least one baculovirus. Injection is preferably performed using a needle that pierces the pupa and administers a volume of solution containing the baculovirus into the body of the pupa. This step can also be automated, the pupae may be placed, for example, in a matrix or array of small pores, and an inoculation device or robot (described below) automatically administers the baculovirus into the pupae. may For example, the pupae may be placed in a matrix (or array) of ostia, and an inoculation device or robot (e.g., with needles on a robotic arm) automatically places needles in the ostia so that the needles pierce the operculum. The matrix has a lid with a hole in the center to access the pupae through the . The needle penetrates the body of the pupa, for example about 1 mm to 5 mm, preferably about 3 mm (which can also be automated), and administers the solution containing the baculovirus inside the pupa. The device or robot is placed in the pupae with a precise amount of baculovirus (eg, about 0.5 containing baculovirus).
A precision pump may be provided that can dispense microliter to 10 microliter volumes of solution, preferably about 5 μl). The robot may comprise an arm with one or more needles capable of injecting baculovirus into pupae at precise locations. Once this has been done, even if the robot is moved out of the ostium, the pupa is easily left in the matrix ostium thanks to the lid of the matrix in which the pupa is placed, and the matrix is closed so that only the needle can pass through the hole. Because the hole in the operculum is smaller than the pupa, the pupa is not removed from the ostium when the robotic arm removes the needle from the pupa, so the needle is removed from the pupa and the ostium matrix, leaving the pupa in the ostium. With this preferred arrangement, the inoculation process is automatic, rapid, efficient and highly reproducible (all pupae receive the same volume of solution containing baculovirus in the same procedure).
. The robot may be mobile and can inoculate pupae with baculovirus at a rate of about 3,000 to about 10,000 pupae per hour. or a needle that can be injected).

For example, each pupa is inoculated with an amount of baculovirus greater than about 50 plaque-forming units (PFU). For example, about 50, about 100, about 500, about 1000, about 5000, about 10000,
about 15000, about 20000, about 25000, about 30000, about 40000, about 5000
Inoculate each pupa with 0, about 60,000, about 75,000 or more PFU (or even about 1,000,000). For example, as already mentioned above, the baculovirus is contained in solution, so that the pupae are injected with a certain amount of solution containing the baculovirus. For example, the solution may be a cell culture medium containing baculovirus. For example, the solution is 1×PB containing baculovirus.
A buffered solution containing baculovirus, such as a solution of S pH 7.4, 1 mM PMSF (a protease inhibitor), and 5 mM DTT. For example, the pupae contain about 0.5 μm
1 μl to 10 μl, such as about 0.5 μl, such as 1 μl to 10 μl, such as about 1 μl, about 2 μl
A solution containing baculovirus in an amount of 1, about 3 μl, about 5 μl, about 7 μl, or about 10 μl can be inoculated. For example, each pupa contains from 0.5 μl to 50 PFU to 1,000,000 PFU.
Receive 10 μl of solution.

Thanks to the automation of inoculation, the robot can deliver about 3000 or more per hour, for example about 30 per hour.
An inoculation of 00 to about 10,000 pupae may be performed. The robot is suitable for delivering fluids to pupae provided in matrices or arrays.

Step (c) of the method of the invention comprises incubating the inoculated pupae of step (b) for a period of time sufficient for expression of the at least one recombinant protein. This incubation step is at least about 72 hours, such as about 72 hours (after inoculation), about 96
hours, about 100 hours, about 125 hours, about 150 hours, about 168 hours or more. Preferably, the incubation time is about 96 hours to 168 hours after inoculation, or about 72 hours to 168 hours after inoculation. The inoculation step is preferably carried out at a temperature of about 22°C to 28°C in an incubator without the need for light or humidity control. The inoculated pupae, preferably placed in the matrix pore, may be left in the matrix pore for the period of time (incubation) described above. In another embodiment, the pupae are removed from the matrix and placed elsewhere, such as in a bag or any recipient capable of transpiring, for the incubation period (incubation is in a gaseous container rather than in a completely closed container). It is preferred that the exchange takes place in a container)
.

One skilled in the art will calculate the required incubation time and incubation conditions for each pupa and each recombinant protein based on previous experiments, depending on the protein to be expressed.

Step (d) of the method of the invention comprises obtaining pupae containing at least one recombinant protein expressed during the incubation step (c). Pupa containing at least one recombinant protein can be harvested from the matrix/array (or incubation site). These pupae containing at least one recombinant protein may be stored (frozen or lyophilized) for later processing. For example, pupae containing at least one recombinant protein may be packaged (preferably under vacuum) in a package. Packages containing pupae may be stored, shipped, and/or further processed.

The invention also provides a package comprising a pupa of the invention, preferably said pupa comprising at least one recombinant protein. Preferably, the pupae are packaged under vacuum, and packages of the present invention thus include vacuum pupae that have been, for example, frozen or freeze-dried to avoid oxidation. This allows for easier handling and longer shelf life.

Alternatively, pupae obtained according to step (d) of the above method may be frozen and stored for further processing. For example, the pupae may be frozen at about -20°C, or at about -80°C immediately after harvesting the pupae until further processing. For example, pupae containing at least one recombinant protein,
It may be frozen, for example, at a temperature of about -20°C to -70°C until the recombinant protein is extracted. Pupa containing the recombinant protein may be cryopreserved for long periods of time, such as for more than a year.

The (optional) step (e) of the method of the invention optionally comprises harvesting at least one recombinant protein. A person skilled in the art knows methods and protocols for harvesting at least one recombinant protein contained in the pupae obtained in step (d). Of course, these methods and/or protocols may depend on the nature of the recombinant protein expressed.

For example, proteins are extracted by homogenizing the pupae (eg, in a homogenizer such as a blender homogenizer or a rotor-stator homogenizer or a homogenizer mixer for at least seconds/minutes), preferably in the presence of a buffer of neutral pH. preferably the buffer contains antioxidants (reducing agents), protease inhibitors and suitable detergents. For example, the buffer contains about 1 mM to 25 mM reducing agent and about 0.01% to 2% (v/v) wash product. Preferably, the buffer further comprises a mixture of protease inhibitors.

After homogenization, the extract is sonicated and/or centrifuged (eg 15000 G-2000
0G) to remove insect debris and may be filtered.

For example, extraction means (methods and protocols for harvesting at least one recombinant protein contained in pupae) may include physical disruption of pupae, centrifugation, tangential flow filtration steps, sonication, chromatographic methods, and/or or including sterile filtration.

In a preferred embodiment, the viscosity of the homogenate can be reduced by incubation (filtration) with diatomaceous earth (eg Celite). A protein precipitation step can also be included.

The extract can be clarified by tangential flow filtration using filters available for those skilled in the art to choose according to the nature of the recombinant protein. The buffer may be changed by the diafiltration process (eg in the same tangential flow filtration device).

The (optional) step (f) of the method of the invention optionally comprises purifying at least one recombinant protein. Those skilled in the art are aware of protein purification techniques. For example, purification can be achieved by chromatographic purification, filtration or ultracentrifugation.

A purified recombinant protein can thus be obtained. A schematic diagram of one embodiment of this method is shown in FIG.

The recombinant protein obtainable by the method of the invention has a high degree of purity and can be used in vaccines, diagnostics, cosmetics or therapeutics.

A method for producing the recombinant baculovirus of the present invention The present invention further comprises:
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) with a bacmid suitable for producing a recombinant baculovirus derived from step (a)
transfecting the pupae of
(c) incubating the inoculated pupae of step (b) for a period of time sufficient to produce recombinant baculovirus;
(d) obtaining pupae containing the recombinant baculovirus;
(e) optionally harvesting the recombinant baculovirus;
(f) optionally purifying the recombinant baculovirus;
A method of producing a recombinant baculovirus is provided, comprising:

The pupae provided in step (a) are preferably silk-free pupae as defined above. The pupae are of the genera Trichoplusia, Rachiprusia, Spodoptera, Heliotis, Manzuka,
Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa, more preferably Trichoplusia ni, Rachiprusia nu, Spodoptera frugiperda, Heliothis • It preferably belongs to the species Virescens, Helicovelpa armigera, Helicovelpa zi, Manzuka sexta, Ascarafa odorata, or Samia cynthia, even more preferably Trichoplusia ni.

In step (b), the pupae of step (a) are transfected with a bacmid vector suitable for producing recombinant baculovirus derived from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV).

This step (b) also comprises step (b) of the method for producing the recombinant protein described above.
) (but instead of inoculating the baculovirus, inoculating the bacmid vector), i.e. automatically or semi-automatically (also described below) using the device of the invention. Therefore, it is sometimes called an “inoculation step”. The device delivers a predetermined amount of solution containing a transfer vector and/or bacmid to be inoculated into the pupa by needle injection as described above into the pupa (preferably a small hole with a tap with a hole). ) may be inoculated.

A bacmid vector can be generated by procedures known to those skilled in the art, for example, by sequentially generating a cloning vector, a donor vector, a transfer vector, and finally a bacmid vector. For example, the generation of bacmid vectors is described in International Publication No. 2
It is described in patent application published as 014/086981.

In a preferred embodiment, the transfer vector and/or bacmid contains proteins IE-1, IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. It comprises or consists of a nucleic acid sequence allowing expression above endogenous levels and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein. These nucleic acid sequences have already been described above. For example, protein IE-1,
Nucleic acid sequences allowing expression of protein IE-0 and/or fragments thereof are
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
is preferably selected from the group consisting of

Furthermore, the promoter driving the expression of the recombinant protein is
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
is preferably selected from the group of nucleic acids comprising

The recombination homologous region (hr) is preferably the sequence (hr1) shown in SEQ ID NO:21. A nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator and an enhancer region is preferably selected from the group comprising SEQ ID NO:15-SEQ ID NO:20.

Preferably, the vector that inoculates the pupae is a bacmid.

Step (c) comprises step (b) for a period of time sufficient to produce recombinant baculovirus.
) inoculated pupae. For example, T. This period for the 2nd pupa can be from about 72 hours to about 7 days depending on the virus dose and temperature. A person skilled in the art can calculate the period of time sufficient for recombinant baculovirus to be produced.

Pupa containing the recombinant baculovirus are then obtained (step (d)). Pupa containing the recombinant baculovirus may be saved for further processing. For example, the pupae may be vacuum packaged to reduce oxidation processes, facilitate their manipulation, and extend safe storage time. For example, pupae containing recombinant baculovirus may be frozen (eg, at -20°C to -80°C) prior to further processing. For example, pupae containing recombinant baculovirus may be lyophilized prior to further processing.

Optionally, the recombinant baculovirus may be harvested and purified (step (e) and step (
f)).

The apparatus of the invention also includes a precision pump, a mobile mechanical arm, and a pupa (preferably a pupa of the invention, wherein said pupa preferably does not contain silk and is of the order Lepidoptera, preferably Trichophyton). Prussia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa or Samia, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis or Helicoverpa, more preferably Trichoplusia・It may belong to the species Di, Rachiprusia nu, Spodoptera frugiperda, Heliothis virescens, Helicovelpa armigera, Helicoverpa zi, Manzuka sexta, Ascarafa odorata, or Samia cynthia. A device (also referred to herein as a robot) is provided, comprising a (movable) needle suitable for injecting a fluid (preferably a solution containing a baculovirus or bacmid vector).

As described in detail above, the pupae may be provided in a matrix (or array) so that the device can easily place the pupae automatically, i. is preferred. The matrix preferably comprises taps with holes that are smaller than the pupae (ie the pupae cannot pass through the holes).

The device defines the position of the needle (and/or the position of the mechanical arm) and/or the distance from the tip (end) of the needle to the pupa and/or the penetration distance of the needle into the pupa and/or inoculates the pupa. liquid(
It may further comprise a computer program for calculating the volume of the solution (preferably containing the baculovirus or bacmid vector). In addition, the device of the present invention reduces the time of inoculation of pupae and/or
Or it may further comprise a computer program that calculates the time between various inoculations. The device may further comprise a camera that defines the position of the needle.

In a preferred embodiment, the fluid injected by the device of the invention contains recombinant baculovirus. In another embodiment, the fluid injected by the device of the invention comprises a bacmid vector suitable for the production of recombinant baculovirus derived from AcMNPV as described herein above.

The device of the invention is suitable for carrying out step (b) of the method of producing the recombinant protein of the invention and step (b) of the method of obtaining the recombinant baculovirus of the invention.

For example, the device of the present invention may be used in a range of about 0.5 μL to about 10.0 μL, such as about 0.5 μL.
, such as about 1 μL to about 10 μL, such as about 1 μL, about 2 μL, about 3 μL, about 5 μL, about 7
It is suitable for injecting (inoculating) pupae with volumes of fluid of μL, or about 10 μL.

For example, a needle provided in the device of the present invention can penetrate the pupa at a distance of about 1 mm to about 4.5 mm, preferably about 3 mm.

For example, the device of the present invention comprises several movable inoculation needles and produces approximately 300 pupae per hour.
Fluids (preferably solutions containing baculovirus or bacmid vectors) can be inoculated at a rate of 0 to 10,000.

For example, the fluid injected by the device of the invention preferably contains baculovirus in an amount of 50 PFU to 10 ⁶ PFU per dose injected into each pupa. For example, the device of the present invention injects (inoculates) an amount of baculovirus greater than about 50, or greater than about 100, or greater than about 500 plaque-forming units (PFU) into each pupa. For example, about 50, about 100, about 300, about 50
0, about 1000, about 5000, about 10000, about 15000, about 20000, about 2500
Each pupa is inoculated with 0, about 30,000, about 40,000, about 50,000, about 100,000, about 500,000 or more PFU, such as about 1,000,000 PFU.

The device of the invention is preferably equipped with a high precision pump that allows the device to inoculate the pupae with a desired volume of fluid (preferably a solution containing a baculovirus or bacmid vector) with high precision.

The device of the invention is suitable for delivering fluids to pupae provided in matrices or arrays.

The apparatus of the invention preferably further comprises a computer program for calculating the time of inoculation of pupae and/or the time between inoculations to be sufficient to administer the liquid containing baculovirus to each pupae.

Preferably, the device of the invention further comprises a camera for defining the position of the needle.

The present invention further provides an apparatus for selecting silk (silk selection apparatus). A schematic diagram of an exemplary silk selection apparatus of the present invention is shown in Figure 3 (semi-automatic apparatus for silk selection from T. ni cocoons).

As explained above, the silk sorting apparatus (also called "silk sorter") of the present invention comprises at least one container containing a silk solution. For example, the container contains hypochlorous acid.
The first container is also preferably equipped with a system for releasing liquid through the rearing module containing the cocoons, which aids in more efficiently dissolving the silk surrounding the pupae. It is preferred to apply the dissolving liquid to the cocoons by compressed air agitation to reduce the time required to dissolve the silk cocoons.

The silk selection apparatus of the present invention preferably further comprises a second container, which is a washing container, containing a solution, such as water, suitable for removing traces of silk and silk solution from the pupae. containing and/or dispersing the solution. Pupae (pupa) are preferably sprayed with a solution (preferably water). There is preferably a system for providing air to the container lid to dry the pupae. Therefore, after washing the pupae, it is preferable to air-dry the pupae. At the end of the process, the pupae are silk-free and ready for infection or storage (refrigeration) until use.

Example 1. T. Production of two pupae Insect larvae were reared in reusable or disposable incubators containing several hundred larvae that developed from egg to pupa over 15-18 days (Figure 1). Thereafter, the whole incubator or the collected pupae are immersed in a solution of sodium hypochlorite at a concentration of 0.1% weight/volume to 5% weight/volume, or the pupae are sprayed with the solution to remove the cocoon. The silk fibers were dissolved. After dissolving the silk for several minutes, the pupae were washed with water to remove traces of hypochlorite. The pupae were then dried and stored at 4°C until baculovirus inoculation. As can be seen in Figure 2, this process is similar to that of T. The lower density of Dylepidopteran silk treats makes them easier for the same manipulations as Bombyx harpoon cocoons. Bombyx harpoon cocoons require manual intervention to release the pupae, whereas T. mori cocoons require manual intervention to release the pupae. The silk is easy to dissolve and is removed by automated or semi-automated procedures, facilitating scale-up acquisition of pupae ready for injection with recombinant baculovirus for recombinant protein production.

Example 2. Dual recombinant baculovirus containing an enhancer sequence (hr1) operably linked to chimeric promoters (p6.9 and p10) and overexpressed IE
-1 and IE-0 transactivators are highly efficient in the production of recombinant proteins in insect pupae. Recombinant protein expression driven under the control of an activated chimeric promoter was obtained using conventional baculovirus and insect pupae (
T. d) compared. A gene encoding recombinant green fluorescent protein (GFP) was cloned into a conventional AcMNPV baculovirus under the control of the polyhedrin promoter by conventional means (Fig. 4A). Another AcMNPV baculovirus modified with a cassette containing the regulatory elements mentioned above (TB expression cassette) was also generated containing the gene encoding GFP (Fig. 4B). Pupae were infected with 50 000 PFU of each baculovirus by an inoculation robot equipped with a precision pump capable of administering microliter volumes of virus inoculum and a robotic arm capable of injecting virus at precise locations (Fig. 5A).
). A pupa was placed in the matrix of the ostium with a lid with a hole in the center of each ostium (Fig. 5B).
An inoculation needle (movable) accessed the pupa through the hole and penetrated the pupa several millimeters to inject the baculovirus (Fig. 5C). 0.5 microliters to 10 microliters (μl
) was administered to the pupae, which were held in the ostium by a lid while the needle was withdrawn. This inoculation robot demonstrated an inoculation rate of at least 3000 pupae per hour, at least six times faster than manual inoculation by those skilled in the art.

After an incubation period of 96-168 hours post-inoculation, the pupae are harvested and treated with a neutral pH buffer containing antioxidants, protease inhibitors, and non-ionic detergents (e.g.
Proteins were extracted with a homogenizer in the presence of 1×PBS pH 7.4+5 mM dithiothreitol (DTT)+1 mM phenylmethylsulfonyl fluoride (PMSF)+0.1% Brij35+0.5% sarkosyl). The extract was centrifuged at 15000g-20000g and filtered. Extracts were analyzed by SDS-PAGE electrophoresis and gels were stained with Coomassie blue (Fig. 6A). The production yield (expressed as milligrams per biomass unit) of GFP protein produced by each baculovirus in infected pupae was calculated by densitometry using the bovine serum albumin (BSA) curve.
This assay is genetically modified with the TB cassette versus that obtained with a conventional baculovirus (i.e., a baculovirus containing, for example, the polyhedrin promoter without any other regulatory elements in the expression cassette). The GFP-producing ability of pupae obtained by the baculovirus was increased by about 5.6-fold (Fig. 6B).

Example 3. T. Expression of Various Proteins by TB-Modified Baculovirus in Pupa Similar to conventional baculoviruses that express genes under the control of the drain promoter (polh),
A corresponding recombinant baculovirus was obtained (Fig. 7). The two genes used to obtain the recombinant baculovirus are porcine circovirus 2 derived from the PCV2a GER3 strain.
Protein Cap (SEQ ID NO: 25 and SEQ ID NO: 27) by type, and virus strain A/PR
SEQ. The resulting TB(-) (ie containing the polyhedrin promoter for protein expression in the expression cassette, but TopB
conventional baculovirus unmodified by the ac(TB) expression cassette) and TB
The (+) baculovirus was transferred to T. Their productivity in double pupae was compared using the same infection and protein extraction protocols used and described in Example 2.

A comparison of production yields (expressed in milligrams per biomass unit) in infected pupae mediated by conventional (TB(−)) baculovirus or TB-modified (TB(+)) baculovirus showed that for both proteins , showed that the expression cassette TB increased the production yield. In the case of porcine circovirus Cap protein, there was a 2.79-fold increase in protein production when pupae were infected with TB-modified baculovirus compared to pupae infected with conventional baculovirus (Figure 8). For HA protein by influenza virus, this increase was 2.04-fold (Fig. 9). These results indicate that the TB cassette is a T. It was confirmed that the production of the recombinant protein in the two pupae was remarkably increased.

Example 4. Baculovirus-infected T. cerevisiae. Production of recombinant proteins in pupae is more efficient than in larvae. Relative expression of the five recombinant proteins in the TB(+) baculovirus in pupae and larvae of D. The proteins expressed were: GFP (SEQ ID NO:23), Cap (SEQ ID NO:25), HA (SEQ ID NO:30), and two rabbit hemorrhagic calicivirus strains (RHDV genogroup 1 and RHDVb; 32 and SEQ ID NO:33
) (Figure 10). 50000 PF for pupae and larvae
were infected with the corresponding TB(+) baculovirus of U. Infected insects (larvae and pupae) were collected 96 hours after infection. SD of soluble protein extracts stained with Coomassie blue
Analyzed by S-PAGE electrophoresis (FIGS. 11A, 12A, 13A and 14A)
. Recombinant proteins were quantified by densitometry using BSA curves and production yields were expressed as milligrams per unit of biomass (Figures 11B, 12B, 13B and 14B). In both cases, pupae expressed higher amounts of recombinant protein than larvae, with an increase ratio of 1.06 to 3.64.

Example 5. T. Production of Recombinant Virus-Like Particles (VLPs) in Two Pupa To demonstrate the production of VLPs in infected pupae, TB(+) baculovirus expressing VP60 protein from the two rabbit calicivirus strains analyzed in Example 4. Protein extracts from pupae infected with M. were processed for VLP purification. Detergent (PBS (0
. 2% Sarkosyl (Sigma
) and 5 mM EDTA (Sigma)), and a protease inhibitor (Roche's Complete
VLPs were isolated from infected pupae 96 hours post-infection by centrifugation in the presence of e™.
was extracted and incubated overnight at 4°C. They were then subjected to DNAse digestion at 37°C for 1 hour.
I (Roche Diagnostics). After an additional centrifugation (2000×g, 5 minutes), the supernatant was subjected to ultracentrifugation (131453×g; 2.5 hours). Vertre the sediment
l (Sigma) and a second ultracentrifugation (131453×g; 2.5 h) was performed. Finally, the pellet was resuspended in 1×PBS and stored at 4° C. until analysis.

The precipitate was analyzed by transmission electron microscopy performed by conventional means. Briefly,
Purified VLPs (approximately 5 μl) were applied to glow-discharged carbon-coated grids for 2 minutes. Samples were negatively stained with 2% (weight/volume) aqueous uranyl acetate. EM 2000 photomicrograph
Recorded by Ex microscope (JEOL, Japan). As shown in Figure 14, VLPs of the expected size and shape corresponding to RHDV were observed, demonstrating corrected folding and self-assembly in baculovirus-infected pupal tissue (Figure 15).
).

Example 6. Infected T. Production of viral inoculum in two pupae Cell lines of Spodoptera frugiperda (Sf21 and Sf9) were incubated with 10% heat-inactivated fetal bovine serum (PAN Biotech GmbH) and gentamicin (50 μg/ml) (PAN Biotech Gm).
bH) in TNMFH medium (PAN Biotech GmbH, Germany) at 27°C. Cell density and viability were determined by trypan blue staining. Cell viability was calculated based on the percentage of viable cells relative to the total number of cells at various times post-infection.

Sf9 cells cultured in suspension were infected in spinner flasks (80 ml culture medium) at a cell density of 2×10 ⁶ cells/ml. Cell viability upon infection is 99% in suspension.
%. Sf9 cells were infected at an infection efficiency of 0.01 to 0.1 (MOI: multiplicity of in
infection) with recombinant baculovirus in vitro. Post-infection 7
The virus inoculum was harvested from the supernatant after centrifugation from 2 hours to 96 hours. Viral titers were calculated by plaque forming unit (pfu) assay and contained ~10 ⁷ viruses per ml.
A virus titer of 10 ⁸ viruses was obtained.

Virus was used at doses ranging from 5×10 ² pfu to 10 ⁴ pfu to challenge T. Two pupae were infected. After 3-7 days, pupae were treated in cell culture medium or treated with DTT and the protease inhibitor PMSF.
Infectious virus was harvested by homogenization in a special PBS buffer containing . The pupal homogenate was centrifuged at 5000×g for 30 minutes to eliminate pupal debris. The virus-containing supernatant can then be sequentially filtered through 0.45 micron and 0.22 micron filters and the resulting virus preparation preserved by mixing with glycerol prior to freezing or lyophilization. did it. T. Viral stocks were titrated in 5th instar larvae of D.
titrated), specifically the LID50 (larvae infective dose) using the Karber method.
ectious dose)50) was identified. The statistical significance of one infectious dose calculated in this way is given by the ID
That is, a population of larvae infected at 50 would represent 50% infected individuals. Larvae were observed for at least 96 hours to detect clinical signs and follow their development to pupae. Typical virus titers range from 10 ⁶ pfu per ml of virus preparation.
10 ⁸ pfu. Additionally, baculovirus inoculum can be obtained without prior generation of baculovirus vectors in cell culture. Bacmids obtained in bacteria by gene transposition procedures can be used to transfect pupae. Figure 16 represents the basic procedure for obtaining virus stocks in pupae by the cell-free system.

Example 7. Cell Cultures and Viruses Spodoptera frugiperda Sf21 or Sf9 cell lines were grown in 6-well tissue culture plates (1×10 ⁶ cells/well) in 10% heat-inactivated fetal bovine serum (Pan, Germany).
in TNM-FH insect medium (Pan Biotech™, Germany) containing
Cultured at 27°C. AcMNPV recombinant baculovirus was obtained by the “Bac-to-Bac™” baculovirus expression system (Invitrogen™, USA). A variety of TB(+) transfer vectors containing recombinant DNA regulatory elements are pFastB
generated using the ac™-DUAL plasmid (Invitrogen™). Cellfectin™ (invitrog, USA) using these transfer vectors
en™) were transfected into Sf21 cells. Recombinant baculoviruses resulting from infection of Sf21 cells were then passaged twice in cells and titered by plaque assay. The resulting TB(+) baculovirus expression cassette gene construct was combined with a combination of gene regulatory elements involved in gene expression (polhAc-ie
-01/hr1p6.9p10, SEQ ID NO: 17, plus Figures 4, 7 and 10 showing the sequence of the gene encoding the desired protein, e.g. is shown schematically in 6, 8, and 8 using various expression cassettes.
Recombinant baculoviruses used in the examples shown in FIGS. 9, 11, 12, 13 and 14 were generated.

Example 8. Generation of cloning vectors Cloning vectors can insert foreign DNA fragments by treating the vehicle and foreign DNA with restriction enzymes that create the same overhangs and then ligating the fragments together, TB(+) baculovirus expression. A small piece of DNA that contains a cassette. The essential features of a cloning vector are the foreign gene in the orientation in which it is chosen, the actual transformed cell in bacteria and a functional origin of replication (ORI) for propagation.
It must contain a synthetic multiple cloning site (MCS) that facilitates the insertion of selectable markers, such as those for antibiotic resistance, to allow selection of .

Example 9. Generation of a Donor Vector Containing the Baculovirus Expression Cassette of the Invention The donor vector consists of a cloning vector, such as the pUC57 plasmid, containing the baculovirus expression cassette into which the foreign gene has been cloned using appropriate restriction enzymes. The TB(+) baculovirus expression cassette used was synthesized by ligation of the following DNA sequences: (i) a sequence downstream of a promoter sequence such as the polh promoter (eg SEQ ID NO: 10) and upstream of the HSV TK polyadenylation signal; Ac-ie-
a baculovirus transcriptional regulator encoding 01 (eg, SEQ ID NOs: 1-5);
and (ii) at another locus following the homology region hr1 upstream of an enhancer sequence, such as (iii) a promoter sequence, such as p6.9p10 (eg SEQ ID NO: 13),
A multiple cloning site (MCS) for cloning the gene of interest and a p10 polyadenylation signal downstream of the MCS (Figures 4, 7 and 10). Baculovirus expression cassettes can be used to facilitate subcloning of commercial baculovirus production systems into transfer vectors (gene transposition, e.g., based on the "Bac-to-Bac™" system (invitrogen™), or homologous recombination, e.g. "flashBAC (
Trademark)" (Oxford Expression Technologies™), "Baculogold™" (BD Biosciences™), "BacPAK6™" (Clontech™),
Based on “Bac-N-Blue DNA™” (invitrogen™), flanked by specific restriction sites (eg Bgl II and BstZ171 at the 5′ end and 3′
Terminal Bgl II and Sgf I).

The coding foreign gene was cloned into the cloning vector MCS using Nco I and Spe I restriction sites to generate the donor plasmid vector.

Example 10. Generation of a transfer vector containing the expression cassette of the baculovirus of the invention Transfer vector was digested with donor vector containing BstZ17 1 and Xba I at the 5' flanking site and transfer vector pF similarly digested with the same enzymes.
It was generated by cloning into astBac™1. In this case, subcloning results in exchange of the SV40 polyadenylation signal of the baculovirus expression cassette with the p10 polyadenylation signal from the transfer vector. Separately, all elements of the expression cassette are contained in the pFastBac transfer vector, replacing the polh promoter and MCS of the original commercial transfer vector.

Example 11. Generation of Baculovirus Expression Vectors Comprising the Baculovirus Expression Cassettes of the Invention Using the "Bac-to-Bac™" System Using the modified transfer vector pFastBac™1 and the TB(+) baculovirus expression cassette, Recombinant baculovirus was generated by using the "Bac-to-Bac™" baculovirus expression system. More specifically, E . E. coli host strain DH10Bac™ was transformed to generate recombinant bacmids following transposition of the expression cassette. Recombinant bacmid DNA containing the TB(+) baculovirus expression cassette of the present invention and various foreign-encoded genes was then used to transfect insect cells, such as Sf21 cells, using Cellfectin™. The bacmid was also used to transfect insect pupae. 1- to 5-day-old Trichoplusia ni pupae were used for this experiment. 72 hours after transfection, cells or pupae were harvested and processed,
Generation of the first recombinant baculovirus was obtained. This recombinant baculovirus could then be further amplified and/or titered according to conventional protocols. Similar procedures can be used to generate recombinant baculovirus with other transfer vectors provided by commercial BEVS.

Example 12. Infection of Insect Pupae One- to five-day-old Trichoplusia ni pupae were used for all experiments. The standard weight of each pupa was approximately 200-300 mg and was diluted in cell culture medium or 1×PBS to reach the selected number of plaque-forming units (PFU) per dose, either manually or by a specially designed robot. 1-10 μl of recombinant baculovirus was injected into pupae. Pupae were collected 72-168 hours after infection. Harvested pupae were immediately frozen and stored at −20° C. or −80° C. until processed for quantification of recombinant protein. Frozen T. cerevisiae infected with baculovirus. Total soluble non-denatured protein (TSNDP
: Total soluble, non-denatured proteins) in the presence of extraction buffer for several minutes.
Obtained by homogenization using a blender or homogenizer mixer.

Example 13. Downstream treatment of insect pupae Frozen pupae were crushed with a homogenizer and treated with a reducing agent, 0.01, at a concentration of 1 mM to 25 mM.
A crude extract containing a mixture of detergents and protease inhibitors at a concentration of % to 2% was obtained. After the viscosity of the crude extract was reduced by its incubation with diatomaceous earth for a period of time, it was centrifuged to remove insect debris and filtered to remove diatomaceous earth. The extract was then clarified by tangential flow filtration using appropriate filters according to the nature of the recombinant protein. Finally, a diafiltration process was performed on the same tangential flow filtration device, changing the buffer before further chromatographic purification. FIG. 17 represents a general procedure for obtaining soluble recombinant protein extracts from baculovirus-infected pupae.

Item (I) of the present invention:
1. Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV
) containing recombinant baculoviruses and/or bacmids.

2. The pupa of paragraph 1, wherein said pupa belongs to the order Lepidoptera.

3. The pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia ni, Manzuka sexta, Spodoptera frugiperda, Spodoptera spp. Litoralis spp., Ascarafa odorata spp., Helicovelpa zi spp., Heliothis virescens spp., Rachiprusia nu spp. or Samia cynthia spp., or AcMNPV
2. A pupa according to paragraph 1, which is any other pupa susceptible to infection with a recombinant baculovirus and/or bacmid derived from .

4. The baculovirus allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein.

5. a nucleic acid sequence that allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection; and a recombinant homology region (hr) operably linked to any suitable promoter to drive expression of the recombinant protein.

6. 6. Pupae according to paragraph 5, wherein said pupae belong to the order Lepidoptera.

7. The pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia ni, Manzuka sexta, Spodoptera frugiperda, Spodoptera spp. Litoralis spp., Ascarafa odorata spp., Helicovelpa zi spp., Heliothis virescens spp., Rachiprusia nu spp. or Samia cynthia spp., or AcMNPV
7. A pupa according to paragraph 6, which is any other pupa susceptible to infection with a recombinant baculovirus and/or bacmid derived from .

8. 8. A pupa according to any one of paragraphs 1 to 7, wherein said pupa belongs to the species Trichoplusia ni.

9. a nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof,
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
9. The pupa according to any one of paragraphs 4 to 8, selected from the group consisting of

10. a promoter driving expression of the recombinant protein,
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
10. A pupa according to any one of paragraphs 4 to 9, selected from the group of nucleic acids comprising

11. The recombination homology region (hr) is the sequence (hr1) shown in SEQ ID NO: 21,
A pupa according to any one of Items 4 to 10.

12. a nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator, and an enhancer region is selected from the group comprising SEQ ID NOs: 15-20;
A pupa according to any one of Items 4 to 11.

13. 13. The pupa of any one of paragraphs 4-12, further comprising a nucleic acid sequence encoding a recombinant protein.

14. The recombinant protein is a subunit monomer vaccine, subunit multimer vaccine, virus-like particle, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone, diagnostic protein reagent , and green fluorescent protein (GF
14. Pupae according to paragraph 13, selected from the group consisting of:

15. The recombinant protein is
(a) a capsid protein from porcine circovirus, preferably porcine circovirus type 2 (e.g. SEQ ID NO: 26);
(b) foot and mouth disease virus VP1 protein, VP3 protein or VP0 protein;
(c) canine parvovirus VP1 and VP2 proteins;
(d) porcine parvovirus VP1 and VP2 proteins;
(e) human norovirus (genogroup I or II) capsid proteins;
(f) a calicivirus capsid protein;
(g) L derived from human papillomavirus, preferably human papillomavirus 16
1 protein,
(h) hepatitis E protein E2;
(i) Infectious bursal disease virus VP1 protein, VP2 protein and V
P3 protein,
(j) an astrovirus ORF2-encoded protein;
(k) influenza virus HA protein (e.g. SEQ ID NO: 30), NA protein and M1 protein;
(l) hepatitis B core antigen and surface antigen;
(m) human parvovirus VP1 and VP2 proteins, and
(n) VP6 derived from rabbit calicivirus, preferably rabbit hemorrhagic disease virus
0 proteins (e.g. SEQ ID NO: 32 and SEQ ID NO: 33),
15. The pupa of Paragraph 14, which is a virus-like particle protein selected from the group consisting of:

16. Use of pupae as defined in any one of paragraphs 1-15 for the expression of recombinant proteins.

17. The recombinant protein is a subunit monomer vaccine, subunit multimer vaccine, virus-like particle, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone, diagnostic protein reagent , and green fluorescent protein (GF
17. Use according to paragraph 16, selected from the group consisting of: P).

18. The recombinant protein is
(a) a capsid protein from porcine circovirus, preferably porcine circovirus type 2 (e.g. SEQ ID NO: 26);
(b) foot and mouth disease virus VP1 protein, VP3 protein or VP0 protein;
(c) canine parvovirus VP1 and VP2 proteins;
(d) porcine parvovirus VP1 and VP2 proteins;
(e) human norovirus (genogroup I or II) capsid proteins;
(f) a calicivirus capsid protein;
(g) L derived from human papillomavirus, preferably human papillomavirus 16
1 protein,
(h) hepatitis E protein E2;
(i) Infectious bursal disease virus VP1 protein, VP2 protein and V
P3 protein,
(j) an astrovirus ORF2-encoded protein;
(k) influenza virus HA protein (e.g. SEQ ID NO: 30), NA protein and M1 protein;
(l) hepatitis B core antigen and surface antigen;
(m) human parvovirus VP1 and VP2 proteins, and
(n) VP6 derived from rabbit calicivirus, preferably rabbit hemorrhagic disease virus
0 proteins (e.g. SEQ ID NO: 32 and SEQ ID NO: 33),
18. Use according to paragraph 17, which is a virus-like particle protein selected from the group consisting of

19.
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) inoculating the pupae of step (a) with a recombinant baculovirus derived from
(c) for a period of time sufficient to express the at least one recombinant protein (
incubating the inoculated pupae of b);
(d) obtaining pupae containing at least one recombinant protein;
(e) optionally harvesting at least one recombinant protein;
(f) optionally purifying at least one recombinant protein;
A method of producing at least one recombinant protein, comprising:

20. 20. The method of paragraph 19, wherein said pupa belongs to the order Lepidoptera.

21. The pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia ni, Manzuka sexta, Spodoptera frugiperda, Spodoptera spp. Any susceptible to recombinant baculoviruses and/or bacmids belonging to Litoralis sp., Ascarafa odorata sp., Helicovelpa zee sp., Heliothis virescens sp., Rachiprusia nu sp. and Samia cynthia sp. or derived from AcMNPV 21. A method according to paragraph 20, which is another pupa of

22. 22. The method of paragraph 21, wherein said pupa belongs to the species Trichoplusia ni.

23. The recombinant baculovirus is capable of expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. and a recombination homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein. .

24. said nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof,
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence encoding a protein having 5% sequence identity and capable of functioning as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
24. The method of Clause 23, selected from the group consisting of

25. the promoter driving expression of the recombinant protein,
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
25. The method of paragraph 23 or 24, wherein the method is selected from the group of nucleic acids comprising

26. The recombination homology region (hr) is the sequence (hr1) shown in SEQ ID NO: 21,
Item 26. The method of any one of Items 23-25.

27. a nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator, and an enhancer region is selected from the group comprising SEQ ID NOs: 15-20;
Item 27. The method of any one of Items 23-26.

28. 28. The method of any one of paragraphs 19-27, wherein said pupa further comprises a nucleic acid sequence encoding a recombinant protein.

29. The recombinant protein is a subunit monomer vaccine, subunit multimer vaccine, virus-like particle, therapeutic protein, antibody, enzyme, cytokine, blood clotting factor, anticoagulant, receptor, hormone, diagnostic protein reagent , and green fluorescent protein (GF
P).

30. The recombinant protein is
(a) a capsid protein from porcine circovirus, preferably porcine circovirus type 2 (e.g. SEQ ID NO: 26);
(b) foot and mouth disease virus VP1 protein, VP3 protein or VP0 protein;
(c) canine parvovirus VP1 and VP2 proteins;
(d) porcine parvovirus VP1 and VP2 proteins;
(e) human norovirus (genogroup I or II) capsid proteins;
(f) a calicivirus capsid protein;
(g) L derived from human papillomavirus, preferably human papillomavirus 16
1 protein,
(h) hepatitis E protein E2;
(i) Infectious bursal disease virus VP1 protein, VP2 protein and V
P3 protein,
(j) an astrovirus ORF2-encoded protein;
(k) influenza virus HA protein (e.g. SEQ ID NO: 30), NA protein and M1 protein;
(l) hepatitis B core antigen and surface antigen;
(m) human parvovirus VP1 and VP2 proteins, and
(n) VP6 derived from rabbit calicivirus, preferably rabbit hemorrhagic disease virus
0 proteins (e.g. SEQ ID NO: 32 and SEQ ID NO: 33),
30. The method of Paragraph 29, wherein the virus-like particle protein is selected from the group consisting of:

31. 31. The method of any one of paragraphs 19-30, wherein said pupa is a silk-free pupa.

32. The silk-free pupae are treated with a solution of hypochlorous acid salt, preferably sodium hypochlorite, by T. 32. A method according to Paragraph 31, which is obtainable by dissolving cocoon silk containing two pupae.

33. 33. The method of any one of paragraphs 19-32, wherein inoculation of said pupae with recombinant baculovirus is performed by injecting said baculovirus into said pupae.

34. 34. The method of any one of paragraphs 19-33, wherein said pupa inoculated in step (b) is a pupa as defined in any one of paragraphs 1-15.

35. 35. The method of any one of paragraphs 19-34, wherein the pupae are inoculated with baculovirus in an amount of 50 PFU to 10 ⁶ PFU per pupae.

36.
(a) preparing a pupa contained in a cocoon;
(b) treating said cocoons containing pupae with a solution of hypochlorous acid salt, preferably sodium hypochlorite;
(c) obtaining sterilized silk-free pupae;
A method for producing silk-free pupae belonging to the order Lepidoptera, comprising:

37. 37. The method of Paragraph 36, wherein said pupae do not belong to the Bombyx mori species.

38. 38. A method according to paragraphs 36 or 37, wherein said pupae belong to the species Trichoplusia ni.

39. 39. The method of any one of paragraphs 36-38, wherein said pupae are provided in a matrix (or array).

40.
(a) preparing a pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) with a bacmid suitable for producing a recombinant baculovirus derived from step (a)
transfecting the pupae of
(c) incubating the inoculated pupae of step (b) for a period of time sufficient to produce recombinant baculovirus;
(d) obtaining pupae containing the recombinant baculovirus;
(e) optionally harvesting the recombinant baculovirus;
(f) optionally purifying the recombinant baculovirus;
A method of producing a recombinant baculovirus, comprising:

41. The method of Paragraph 39, wherein said pupa is a pupa as defined in any one of Paragraphs 36-39.

42. 42. The method of paragraph 40 or 41, wherein said pupa belongs to the order Lepidoptera.

43. The pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, preferably Trichoplusia ni, Manzuka sexta, Spodoptera frugiperda, Spodoptera spp. Litoralis spp., Ascarafa odorata spp., Helicovelpa zi spp., Heliothis virescens spp., Rachiprusia nu spp. or Samia cynthia spp., or AcMNP
43. A method according to paragraph 42 which is a recombinant baculovirus derived from V. and/or any other pupae susceptible to infection with bacmid.

44. 44. The method of Paragraph 43, wherein said pupa belongs to the species Trichoplusia ni.

45. Autographa californica multicapsid nuclear polyhedrosis virus (AcMNP
V) the bacmid suitable for the production of recombinant baculovirus derived from protein I, which functions as a transcriptional regulator above endogenous levels obtained during baculovirus infection
Nucleic acid sequences allowing expression above endogenous levels of E-1, protein IE-0 and/or fragments thereof, operably linked to any promoter suitable to drive expression of the recombinant protein. 45. A method according to any one of paragraphs 40 to 44, comprising or consisting of a recombination homology region (hr).

46. said nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof,
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence encoding a protein having 5% sequence identity and capable of functioning as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
46. The method of Paragraph 45, selected from the group consisting of:

47. the promoter driving expression of the recombinant protein,
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
47. The method of paragraph 45 or 46, wherein the method is selected from the group of nucleic acids comprising

48. The recombination homology region (hr) is the sequence (hr1) shown in SEQ ID NO: 21,
48. The method of any one of paragraphs 45-47.

49. a nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator, and an enhancer region is selected from the group comprising SEQ ID NOs: 15-20;
49. The method of any one of paragraphs 45-48.

50. 50. The method of any one of paragraphs 40-49, wherein said pupa is a silk-free pupa.

51. The silk-free pupae are treated with a solution of hypochlorous acid salt, preferably sodium hypochlorite, by T. 51. A method according to Paragraph 50, obtainable by dissolving a cocoon containing two pupae.

52. 52. The method of any one of paragraphs 40-51, wherein said transfected pupa of step (b) is a pupa as defined in any one of paragraphs 5-15.

53. 53. The method of any one of paragraphs 40-52, wherein said pupae are provided in a matrix.

54. precision pump, mobile mechanical arm, Lepidoptera, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, more preferably Trichoplusia spp. Manzuka sexta species, Spodoptera frugiperda species, Spodoptera litoralis species, Ascarafa odorata species, Helicoverpa zi species, Heliothis virescens species, Rakiprusia nu species,
or pupae belonging to the species Samia Cynthia, or any other pupae susceptible to infection with recombinant baculoviruses and/or bacmids derived from AcMNPV (
a movable) needle.

55. 55. Apparatus according to paragraph 54, wherein said pupae are provided in a matrix or array.

56. the device defines the position of the needle and/or calculates the distance from the needle to the pupa and/or the penetration distance of the needle into the pupa and/or the volume of liquid to inoculate the pupa, and 56. Apparatus according to paragraphs 54 or 55, further comprising a computer program for determining the coordinates of the inoculation needle and/or calculating the volume and/or time of inoculation of the pupa and/or the time between inoculations.

57. 57. Apparatus according to paragraph 56, further comprising a computer program for calculating inoculation time and/or time between inoculations of said pupae.

58. 58. The device of any one of clauses 54-57, wherein the device further comprises a camera for defining the position of the needle.

59. 55. The apparatus of Clause 54, wherein said pupa is a pupa as defined in any one of Clauses 1-15.

60. Paragraphs 54-59, wherein said injected fluid comprises recombinant baculovirus or bacmid
A device according to any one of the preceding claims.

61. 61. Apparatus according to any one of clauses 54-60, wherein said apparatus is suitable for performing step (b) as defined in any one of clauses 19 and/or 40.

62. 62. Apparatus according to any one of paragraphs 54-61, wherein the fluid injected into said pupa ranges from about 0.5 μL to about 10.0 μL, preferably about 5 μL.

63. 63. The device of any one of paragraphs 54-62, wherein the needle penetrates the pupa at a distance of about 1 mm to about 5 mm, preferably about 3 mm.

64. The fluid injected into the pupae is preferably from 50 PFU per dose injected into each pupae.
64. The device of any one of paragraphs 54-63, comprising baculovirus in an amount of 10 ⁶ PFU.

Item (II) of the present invention:
1. Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV
), preferably said pupa comprising a recombinant baculovirus and/or bacmid derived from the or Samia, more preferably Trichoplusia ni spp., Manzuka sexta spp., Spodoptera frugiperda spp., Spodoptera litoralis spp., Ascarafa odorata spp., Helicovelpa zi spp., Heliothis virescens spp. A pupa belonging to the species Nunu, or Samia cynthia, or any other pupa susceptible to infection with recombinant baculoviruses and/or bacmids derived from AcMNPV.

2. The baculovirus allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection. and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein.

3. a nucleic acid sequence that allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection; and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein, preferably said pupa is of the order Lepidoptera, preferably of the genus Trichoplusia, genus Rachiprusia,
Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa or Samia, more preferably Trichoplusia ni spp., Manzuka sexta spp., Spodoptera frugiperda spp., Spodoptera litoralis spp., Ascarafa odorata spp. , Helicovelpa zi sp., Heliothis virescens sp., Rachiprusia nu sp., or Samia cynthia sp., or any other pupa susceptible to infection with recombinant baculoviruses and/or bacmids derived from AcMNPV. .

4. a nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof,
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
is selected from the group consisting of
a promoter driving expression of the recombinant protein,
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
selected from the group of nucleic acids comprising
4. A pupa according to Item 2 or 3, wherein said recombination homology region (hr) is the sequence (hr1) shown in SEQ ID NO:21.

5. 5. The method according to any one of items 2 to 4, wherein the nucleic acid sequence comprising the combination of the recombinant promoter, transcription regulatory factor-encoding sequence, and enhancer region is selected from the group comprising SEQ ID NO: 15 to SEQ ID NO: 20. Pupae as described.

6. The pupae are preferably subunit monomer vaccines, subunit multimer vaccines,
A recombinant protein selected from the group consisting of virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP). 6. The pupa of any one of paragraphs 2-5, further comprising an encoding nucleic acid sequence.

7. Use of pupae as defined in any one of paragraphs 1 to 6, preferably subunit monomeric vaccines, subunit multimeric vaccines, virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood Use of pupae for expression of recombinant proteins selected from the group consisting of clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP).

8.
(a) providing a pupa, preferably a silk-free pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) inoculating the pupae of step (a) with a recombinant baculovirus derived from
(c) for a period of time sufficient to express the at least one recombinant protein (
incubating the inoculated pupae of b);
(d) obtaining pupae containing at least one recombinant protein;
(e) optionally harvesting at least one recombinant protein;
(f) optionally purifying at least one recombinant protein;
including
Preferably, said pupa is of the order Lepidoptera, preferably of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, even more preferably Trichoplusia ni, Manzuka Sexta species, Spodoptera
Recombinant baculoviruses and/or bacmids belonging to Frugiperda sp., Spodoptera litoralis sp., Ascarafa odorata sp., Helicovelpa zi sp., Heliothis virescens sp., Rachiprusia nu sp. or Samia cynthia sp. or derived from AcMNPV A method of producing at least one recombinant protein that is any other pupae susceptible to infection with .

9. protein IE-1, protein IE-0 and/or wherein said recombinant baculovirus functions as a transcriptional regulator above endogenous levels obtained during baculovirus infection
or a nucleic acid sequence that allows expression above endogenous levels of these fragments and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein; Preferably, said protein IE-1, protein I, which functions as a transcriptional regulator above endogenous levels obtained during baculovirus infection
The above nucleic acid sequences allowing expression above endogenous levels of E-0 and/or fragments thereof, and the above recombinant homology regions operably linked to any promoter suitable for driving expression of the recombinant protein. 9. The method of claim 8, wherein (hr) is as defined in claim 4.

10. a nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator, and an enhancer region is selected from the group comprising SEQ ID NOs: 15-20;
Item 12. The method according to Item 11.

11. The pupae are preferably subunit monomeric vaccines, subunit multimeric vaccines, virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents and green fluorescent protein (GFP)
Sections 8 to 8, further comprising a nucleic acid sequence encoding a recombinant protein selected from the group consisting of
11. The method of any one of 10.

12.
(a) preparing a pupa contained in a silk cocoon;
(b) treating said silk cocoons containing pupae with a solution of hypochlorous acid salt, preferably sodium hypochlorite;
(c) obtaining sterilized silk-free pupae;
Lepidoptera, preferably T. A method for producing silk-free pupae belonging to two species.

13.
(a) preferably Lepidoptera, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, even more preferably Trichoplusia ni, Manzuka sexta Spodoptera frugiperda spp., Spodoptera litoralis spp., Ascarafa odorata spp., Helicovelpa zi spp., Heliothis virescens spp., Rachiprusia nu spp. or Samia cynthia spp. providing any other pupae susceptible to virus and/or bacmid infection, preferably silk-free pupae;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
transfecting the pupae of step (a) with a bacmid suitable for the production of recombinant baculovirus derived from PV);
(c) incubating the inoculated pupae of step (b) for a period of time sufficient to produce recombinant baculovirus;
(d) obtaining pupae containing the recombinant baculovirus;
(e) optionally harvesting the recombinant baculovirus;
(f) optionally purifying the recombinant baculovirus;
A method of producing a recombinant baculovirus, comprising:

14. Autographa californica multicapsid nuclear polyhedrosis virus (AcMNP
V) the bacmids suitable for producing recombinant baculoviruses derived from V) are proteins IE-1, IE-0 and/or proteins functioning as transcriptional regulators above endogenous levels obtained during baculovirus infection. or a nucleic acid sequence that allows expression above endogenous levels of these fragments and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein; or consist of them, preferably above endogenous levels of said protein IE-1, protein IE-0 and/or fragments thereof which function as transcription regulators above endogenous levels obtained during baculovirus infection said nucleic acid sequence enabling expression and said recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein as defined in claim 4 14. The method according to Item 13.

15. a nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator, and an enhancer region is selected from the group comprising SEQ ID NOs: 15-20;
Item 15. The method according to Item 14.

16. precision pump, mobile mechanical arm, Lepidoptera, preferably Trichoplusia, Rachiprusia, Spodoptera, Heliothis, Manzuka, Helicoverpa, Ascarafa, or Samia, even more preferably Trichoplusia genus , Manzuka sexta, Spodoptera frugiperda, Spodoptera litoralis, Ascarafa
pupae belonging to the species Odorata, Helicovelpa zi, Heliothis virescens, Rachiprusia nu, or Samia cynthia, or any other pupae susceptible to infection with recombinant baculoviruses and/or bacmids derived from AcMNPV. and (movable) needles suitable for injecting fluid, preferably the pupae are provided in a matrix or array.

17. The device defines the position of the needle and/or the distance from the needle to the pupa and/or the penetration distance of the needle into the pupa and/or the liquid with which the pupa is inoculated (preferably a recombinant baculovirus or said apparatus preferably for determining the coordinates of said inoculation needle and/or said inoculation volume and/or time and/or inoculation of said pupae. 17. Apparatus according to clause 16, further comprising a computer program for calculating the time between.

Claims (19)

Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)
, wherein the pupa comprises a recombinant baculovirus and/or bacmid derived from C., said pupa belonging to the genera Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa. Said recombinant baculovirus and/or bacmid comprises a nucleic acid sequence encoding a recombinant protein, said recombinant protein preferably being a subunit monomeric vaccine, a subunit multimeric vaccine, a virus-like particle, a therapeutic protein, an antibody, an enzyme , cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP); 2. A pupa according to claim 1, which is not a protein produced in a pupa. protein IE-1, protein IE- wherein said baculovirus and/or bacmid functions as a transcriptional regulator above endogenous levels obtained during baculovirus infection
and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein. including
said nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof,
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
3. A pupa according to claim 1 or 2, selected from the group consisting of: a nucleic acid sequence that allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection; and a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein,
The pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis or Helicovelpa, preferably Trichoplusia ni spp., Spodoptera frugiperda spp., Spodoptera litoralis spp.・Belongs to the Nu species,
a nucleic acid sequence enabling expression of said protein IE-1, protein IE-0 and/or fragments thereof comprising
(a) a nucleic acid comprising a nucleotide sequence set forth in any of SEQ ID NO: 1 to SEQ ID NO: 5;
(b) a nucleotide sequence set forth in any of SEQ ID NOs: 1-5 and at least 7
0%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 9
a nucleic acid sequence that has 5% sequence identity and encodes a protein that can function as a transcriptional regulator in a recombinant baculovirus;
(c) a nucleic acid sequence encoding an amino acid comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, and
(d) at least 70% of the amino acid sequence shown in any of SEQ ID NO: 6 to SEQ ID NO: 9
, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%
a nucleic acid sequence encoding an amino acid sequence that has the sequence similarity of and can function as a transcriptional regulator in a recombinant baculovirus;
A pupa selected from the group consisting of a promoter driving expression of the recombinant protein,
(a) shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to
a nucleic acid comprising the nucleotide sequence shown in any of SEQ ID NO: 13, and
(b) can function as a promoter in a recombinant baculovirus and is shown in any of SEQ ID NO: 10 to SEQ ID NO: 14, preferably SEQ ID NO: 11 to SEQ ID NO: 1
3 and at least 70%, preferably at least 7
a nucleic acid sequence having a sequence identity of 5%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;
and/or
wherein the recombination homology region (hr) is the sequence (hr1) shown in SEQ ID NO: 21;
A pupa according to any one of claims 2-4. 6. The nucleic acid sequence according to any one of claims 2-5, wherein the nucleic acid sequence comprising the combination of a recombinant promoter, a sequence encoding a transcriptional regulator and an enhancer region is selected from the group comprising SEQ ID NO: 15-SEQ ID NO: 20. Pupae as described. Said pupae are preferably subunit monomeric vaccines, subunit multimeric vaccines, virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic proteins Claims 3-6, further comprising a reagent and a nucleic acid sequence encoding a recombinant protein selected from the group consisting of green fluorescent protein (GFP).
A pupa according to any one of claims 1 to 3. Use of pupae as defined in any one of claims 1 to 7, preferably subunit monomeric vaccines, subunit multimeric vaccines, virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, for the expression of a recombinant protein selected from the group consisting of blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP), and/or said recombinant protein is The use preferably is not a protein endogenously produced by the pupa. (a) providing a pupa, preferably a silk-free pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) inoculating the pupae of step (a) with a recombinant baculovirus derived from
(c) for a period of time sufficient to express the at least one recombinant protein (
incubating the inoculated pupae of b);
(d) obtaining pupae containing at least one recombinant protein;
(e) optionally harvesting at least one recombinant protein;
(f) optionally purifying at least one recombinant protein;
including
said pupa is of the genus Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa, preferably Trichoplusia ni, Spodoptera frugiperda, Spodoptera litoralis, Helicovelpa zi, Heliothis virescens, Or a method of producing at least one recombinant protein belonging to the Rachiprusia nu species. The recombinant baculovirus is
a nucleic acid sequence that allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection;
a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein;
including
Said nucleic acid sequence allowing expression above endogenous levels of said protein IE-1, protein IE-0 and/or fragments thereof functioning as transcriptional regulators above endogenous levels obtained during baculovirus infection. , and said recombinant homology region (hr
) is as defined in claims 3 and/or 4. said nucleic acid sequence comprising a combination of a recombinant promoter, a sequence encoding a transcriptional regulator and an enhancer region is selected from the group comprising SEQ ID NO: 15 to SEQ ID NO: 20;
11. The method of claim 10. The pupae are preferably subunit monomer vaccines, subunit multimer vaccines,
A recombinant protein selected from the group consisting of virus-like particles, therapeutic proteins, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents, and green fluorescent protein (GFP). A method according to any one of claims 9 to 11, further comprising a coding nucleic acid sequence and/or said recombinant protein is preferably not a protein endogenously produced by the pupa. (a) preparing a pupa contained in a silk cocoon;
(b) treating said silk cocoons containing pupae with a solution of a hypochlorous acid salt, preferably sodium hypochlorite;
(c) obtaining silk-free, externally sterilized pupae;
Lepidoptera, preferably T. A method for producing silk-free pupae belonging to two species. (a) Trichoplusia, Rachiprusia, Spodoptera, Heliothis, or Helicovelpa, preferably Trichoplusia ni, Spodoptera frugiperda, Spodoptera litoralis, Helicovelpa zi, Heliothis virescens, or providing a pupa belonging to the species Rachiprusia nu, preferably a silk-free pupa;
(b) Autographa californica multicapsid nuclear polyhedrosis virus (AcMN)
PV) with a bacmid suitable for producing a recombinant baculovirus derived from step (a)
transfecting the pupae of
(c) incubating the inoculated pupae of step (b) for a period of time sufficient to produce recombinant baculovirus;
(d) obtaining pupae containing said recombinant baculovirus;
(e) optionally harvesting the recombinant baculovirus;
(f) optionally purifying said recombinant baculovirus;
A method of producing a recombinant baculovirus, comprising: Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)
said bacmid suitable for producing a recombinant baculovirus derived from
a nucleic acid sequence that allows expression above endogenous levels of proteins IE-1, protein IE-0 and/or fragments thereof that function as transcriptional regulators above endogenous levels obtained during baculovirus infection;
a recombinant homology region (hr) operably linked to any promoter suitable for driving expression of the recombinant protein;
comprising or consisting of
Said nucleic acid sequence allowing expression above endogenous levels of said protein IE-1, protein IE-0 and/or fragments thereof functioning as transcriptional regulators above endogenous levels obtained during baculovirus infection. , and said recombinant homology region (hr
) is as defined in claims 3 and/or 4. 16. The method of claim 15, wherein the nucleic acid sequence comprising the combination of the recombinant promoter, the sequence encoding the transcriptional regulator and the enhancer region is selected from the group comprising SEQ ID NO:15-SEQ ID NO:20. precision pump, mobile mechanical arm, Trichoplusia, Rachiprusia, Spodoptera, Heliothis or Helicovelpa, preferably Trichoplusia ni sp., Spodoptera frugiperda sp., Spodoptera litoralis sp., Helicovelpa jii sp., Heliotis • A device comprising a (movable) needle suitable for injecting fluid into pupae belonging to the species Virescens or Rachiprusia nu, preferably provided in a matrix or array. The device defines the position of the needle and/or the distance from the needle to the pupa and/or
or further comprising a computer program for calculating the needle penetration distance into said pupa and/or the volume of liquid (preferably containing recombinant baculovirus or bacmid) to inoculate said pupa,
17. The device preferably further comprises a computer program, for example determining the coordinates of the inoculation needle and/or calculating the volume and/or the time of inoculation of the pupae and/or the time between inoculations. The apparatus described in . A silk selection device comprising at least one container containing a silk lysate, said device being preferably a washing container, containing a solution suitable for removing traces of silk and silk lysate from pupae, and /or Silk selection apparatus comprising a second container for dispersing said solution and optionally a system for providing air for drying the pupae.

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